Substituted pyrido[3,2-d]pyrimidines capable of inhibiting tyrosine kinases of the epidermal growth factor receptor family

ABSTRACT

Novel 4-substituted amino pyrido [3,2-d]pyrimidine inhibitors of epidermal growth factor receptor family of tyrosine kinases are described, as well as pharmaceutical compositions of the same, which are useful in treating proliferative diseases such as cancer, synovial pannus invasion in arthritis, psoriasis, vascular restenosis and angiogenesis and additionally useful in the treatment of pancreatitis and kidney disease as well as a contraceptive agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.08/358,351, filed Dec. 23, 1994, now U.S. Pat. No. 5,654,307 which is acontinuation-in-part of application Ser. No. 08/186,745, filed Jan. 25,1994 and application Ser. No. 08/186,735, filed Jan. 25, 1994 abandoned.

TECHNICAL FIELD

The present invention relates to bicyclic heteroaromatic compounds whichinhibit the epidermal growth factor receptor and related receptors and,in particular, their tyrosine kinase enzymic activity.

BACKGROUND ART

Cancer is generally a disease of the intracellular signalling system, orsignal transduction mechanism. Cells receive instructions from manyextracellular sources, instructing them to either proliferate or not toproliferate. The purpose of the signal transduction system is to receivethese and other signals at the cell surface, get them into the cell, andthen pass the signals on to the nucleus, the cytoskeleton, and transportand protein syntheses machinery. The most common cause of cancer is aseries of defects, either in these proteins, when they are mutated, orin the regulation of the quantity of the protein in the cell such thatit is over or under produced. Most often, there are key lesions in thecell which lead to a constitutive state whereby the cell nucleusreceives a signal to proliferate, when this signal is not actuallypresent. This can occur through a variety of mechanisms. Sometimes thecell may start to produce an authentic growth factor for its ownreceptors when it should not, the so-called autocrine loop mechanism.Mutations to the cell surface receptors, which usually signal into thecell by means of tyrosine kinases, can lead to activation of the kinasein the absence of ligand, and passing of a signal which is not reallythere. Alternatively, many surface kinases can be overexpressed on thecell surface leading to an inappropriately strong response to a weaksignal. There are many levels inside the cell at which mutation oroverexpression can lead to the same spurious signal arising in the cell,and there are many other kinds of signalling defect involved in cancer.This invention touches upon cancers which are driven by the threemechanisms just described, and which involve cell surface receptors ofthe epidermal growth factor receptor tyrosine kinase family (EGFR). Thisfamily consists of the EGF receptor (also known as Erb-B1), the Erb-B2receptor, and its constituitively active oncoprotein mutant Neu, theErb-B3 receptor and the Erb-B4 receptor. Additionally, other biologicalprocesses driven through members of the EGF family of receptors can alsobe treated by compounds of the invention described below.

The EGFR has as its two most important ligands Epidermal Growth Factor(EGF) and Transforming Growth Factor alpha (TGFalpha). The receptorsappear to have only minor functions in adult humans, but are apparentlyimplicated in the disease process of a large portion of all cancers,especially colon and breast cancer. The closely related Erb-B2 Erb-B3and Erb-B4 receptors have a family of Heregulins as their major ligands,and receptor overexpression and mutation have been unequivocallydemonstrated as the major risk factor in poor prognosis breast cancer.Additionally, it has been demonstrated that all four of the members ofthis family of receptors can form heterodimeric signalling complexeswith other members of the family, and that this can lead to synergistictransforming capacity if more than one member of the family isoverexpressed in a malignancy. Overexpression of more than one familymember has been shown to be relatively common in human malignancies.

The proliferative skin disease psoriasis has no good cure at present. Itis often treated by anticancer agents such as methotrexate, which havevery serious side effects, and which are not very effective at thetoxicity-limited doses which have to be used. It is believed thatTGFalpha is the major growth factor overproduced in psoriasis, since 50%of transgenic mice which overexpress TGF alpha develop psoriasis. Thissuggests that a good inhibitor of EGFR signalling could be used as anantipsoriatic agent, preferably, but not necessarily, by topical dosing.

EGF is a potent mitogen for renal tubule cells. Fourfold increases inboth EGF urinary secretion and EGF MRNA have been noted in mice withearly stage streptozoicin-induced diabetes. In addition increasedexpression of the EGFR has been noted in patients with proliferativeglomerulonephritis (Roychaudhury et al. Pathology 1993, 25, 327). Thecompounds of the current invention should be useful in treating bothproliferative glomerulonephritis and diabetes-induced renal disease.

Chronic pancreatitis in patients has been reported to correlate withlarge increases in expression for both EGFR and TGF alpha. (Korc et al.Gut 1994, 35, 1468). In patients showing a more severe form of thedisease, typified by an enlargement of the head of the pancreas, therewas also shown to be overexpression of the erb-B2 receptor (Friess etal. Ann. Surg. 1994, 220, 183). The compounds of the current inventionshould prove useful in the treatment of pancreatitis.

In the processes of blastocyte maturation, blastocyte implantation intothe uterine endometrium, and other periimplantation events, uterinetissues produce EGF and TGF alpha (Taga Nippon Sanka Fujinka GakkaiZasshi 1992, 44, 939), have elevated levels of EGFR (Brown et al.Endocrinology, 1989, 124, 2882), and may well be induced to produceheparin-binding EGF by the proximity of the developing, but notarrested, blastocyte (Das et al. Development 1994, 120, 1071). In turnthe blastocyte has quite a high level of TGF alpha and EGFR expression(Adamson Mol. Reprod. Dev. 1990, 27, 16). Surgical removal of thesubmandibular glands, the major site of EGF secretion in the body, andtreatment with anti-EGFR monoclonal antibodies both greatly reducefertility in mice (Tsutsumi et al. J. Endocrinology 1993, 138, 437), byreducing successful blastocyte implantation. Therefore, compounds of thecurrent invention should prove to have useful contraceptive properties.

PCT patent application Nos. WO92/07844 published May 14, 1992 andWO92/14716 published Sep. 3, 1992 describe 2,4-diaminoquinazoline aspotentiators of chemotherapeutic agents in the treatment of cancer.

PCT published application No. WO92/20642 published Nov. 26, 1992discloses bismono- and bicyclic aryl and heteroaryl compounds whichinhibit EGF and/or PDGF receptor tyrosine kinase.

It is an object of the present invention to inhibit the mitogeniceffects of epidermal growth factor utilizing an effective amount ofbicyclic pyrimidine derivatives, in particular fused heterocyclicpyrimidine derivatives.

It is another object of the present invention to describe bicyclicpyrimidine derivatives, in particular fused heterocyclic pyrimidinederivatives, as inhibitors of the EGF, Erb-B2 and Erb-B4 receptortyrosine kinases.

It is yet another object of the present invention to describe bicyclicpyrimidine derivatives, in particular fused heterocyclic pyrimidinederivatives, that are useful at low dosages as inhibitors of EGF-inducedmitogenesis. This therefore leads to a further object of compoundshaving extremely low cytotoxicity.

It is a further object of the present invention to describe bicyclicpyrimidine derivatives, in particular fused heterocyclic pyrimidinederivatives, that are useful in suppressing tumors, especially breastcancers, where mitogenesis is heavily driven by EGFR family members.

It is another object of the present invention to describe bicyclicpyrimidine derivatives, in particular fused heterocyclic pyrimidinederivatives, that have utility as chronic therapy as inhibitors ofEGF-induced responses.

It is another object of the current invention to describe bicyclicpyrimidine derivatives, in particular fused heterocyclic pyrimidinederivatives, that have utility as therapeutic agents againstproliferative overgrowth diseases, including but not limited to,synovial pannus invasion in arthritis, vascular restenosis, psoriasisand angiogenesis. The compounds disclosed herein also are useful totreat pancreatitis and kidney disease and as a contraceptive agent.

SUMMARY OF THE INVENTION

Described is a method to inhibit epidermal growth factor by treating,with an effective inhibiting amount, a mammal, in need thereof, acompound of Formula I: ##STR1## wherein at least one, and as many asthree of A-E are nitrogen, with the remaining atom(s) carbon, or any twocontiguous positions in A-E taken together can be a single heteroatom,N, O or S, in which case one of the two remaining atoms must be carbon,and the other can be either carbon or nitrogen;

X=O, S, NH or NR⁷, such that R⁷ =lower alkyl (1-4 carbon atoms), OH,NH₂, lower alkoxy (1-4 carbon atoms) or lower monoalkylamino (1-4 carbonatoms);

n=0, 1, 2;

R¹ =H or lower alkyl (1-4 carbon atoms); if n=2, R¹ can be independentlyH or lower alkyl (1-4 carbon atoms) on either linking carbon atom;

R² is lower alkyl (1-4 carbon atoms), cycloalkyl (3-8 carbon atoms),lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms), nitro,halo (fluoro, chloro, bromo, iodo), lower perfluoroalkyl (1-4 carbonatoms), hydroxy, lower acyloxy (1-4 carbon atoms; --O--C(O)R), amino,lower mono or dialkylamino (1-4 carbon atoms), lower mono ordicycloalkylamino (3-8 carbon atoms), hydroxymethyl, lower acyl (1-4carbon atoms; --C(O)R), cyano, lower thioalkyl (1-4 carbon atoms),lower-sulfinylalkyl (1-4 carbon atoms), lower sulfonylalkyl (1-4 carbonatoms), thiocycloalkyl (3-8 carbon atoms), sulfinylcycloalkyl (3-8carbon atoms), sulfonylcycloalkyl (3-8 carbon atoms), sulfonamido, lowermono or dialkylsulfonamido (1-4 carbon atoms), mono ordicycloalkylsulfonamido (3-8 carbon atoms), mercapto, carboxy,carboxamido (--C(O)--NH₂), lower mono or dialkylcarboxamido (1-4 carbonatoms), mono or dicycloalkylcarboxamido (3-8 carbon atoms), loweralkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbonatoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbonatoms), lower alkynyl (2-4 carbon atoms), or two R² taken together oncontiguous carbon atoms can form a carbocyclic ring of 5-7 members or amonounsaturated 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino or thiomorpholino ring; and

m=0-3, wherein Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl,pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl andquinazolinyl;

R³, R⁴, R⁵ and R⁶ are independently, not present, H, lower alkyl (1-4carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy (1-4 carbonatoms), cycloalkoxy (3-8 carbon atoms), hydroxy, lower acyloxy (1-4carbon atoms), amino, lower mono or dialkylamino (1-4 carbon atoms),lower mono or dicycloalkylamino (3-8 carbon atoms), lower alkyl (1-4carbon atoms) or cycloalkyl (3-8 carbon atoms), carbonato (--OC(O)OR)where the R is lower alkyl of 1 to 4 carbon atoms or cycloalkyl of 3-8carbon atoms;

or ureido or thioureido or N- or O- linked urethane any one of which isoptionally substituted by mono or di-lower alkyl (1-4 carbon atoms) orcycloalkyl (3-8 carbon atoms);

lower thioalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbon atoms),mercapto, lower alkenyl (2-4 carbon atoms), hydrazino, N'-loweralkylhydrazino (1-4 carbon atoms), lower acylamino (1-4 carbon atoms),hydroxylamino, lower O- alkylhydroxylamino (1-4 carbon atoms);

or any two of R³ -R⁶ taken together on contiguous carbon atoms can forma carbocyclic ring of 5-7 members or a monounsaturated 1,3-dioxolanyl,1,4-dioxanyl, 1,4-dioxepinyl, pyranyl, furanyl, pyrrolidyl, piperidinyl,thiolanyl, oxazolanyl, thiazolanyl, diazolanyl, piperazinyl, morpholinoor thiomorpholino ring;

any lower alkyl group substituent on any of the substituents in R³ -R⁶which contain such a moiety can be optionally substituted with one ormore of hydroxy, amino, lower monoalkylamino, lower dialkylamino,N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino,N-thiomorpholino or N-piperazino groups;

if one or more of A through E are N, then any of R³ -R⁶ on a neighboringC atom to one of the N atoms, cannot be either OH or SH; and

if any of the substituents R¹, R², R³, R⁴, R⁵ or R⁶ contain chiralcenters, or in the case of R¹ create chiral centers on the linkingatoms, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included.

Described also is a method to inhibit epidermal growth factor bytreating, with an effective inhibiting amount, a mammal, in needthereof, a compound of Formula II: ##STR2## wherein Ar, n, m, R₁ -R₇ andX are the same as in Formula I;

R⁸ is alkyl of from 1-4 carbon atoms or amino or mono or dilower alkyl(1-4 carbon atoms) amino.

The invention is also applicable to the compositions of Formulae I andII with the proviso that at least one of the R³ -R⁶ substituents must betaken singly as a substituent other than hydrogen, halo, lower alkyl(1-4 carbon atoms) or lower alkoxy (1-4 carbon atoms), and with theproviso that A, B, D and E must all be taken singly as carbon ornitrogen atoms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an effect of Examples 6 and 7 on EGF receptorautophosphorylation in A431 human epidermoid carcinoma;

FIG. 2 is an effect of Example 8 on EGF receptor autophosphorylation inA431 human epidermoid carcinoma;

FIG. 3 is a time course for the inhibition of EGF receptorautophosphorylation in A431 by Example 27;

FIG. 4 is an effect of Example 27 on EGF receptor autophosphorylation inA431 cells;

FIG. 5 is an inhibition of EGF receptor autophosphorylation in A431human epidermoid carcinoma by Example 40;

FIG. 6 is an effect of Example 40 on growth factor-mediated tyrosinephosphorylation in Swiss 3T3;

FIG. 7 is an effect of Example 40 on growth factor dependent expressionof c-jun mRNA in Swiss 3T3 mouse fibroblasts;

FIG. 8 is an effect of Example 40 on growth factor mediated expressionof p39^(c-jun) ;

FIG. 9 is an effect of Example 59 of EGF receptor autophosphorylation inA431 human epidermoid carcinoma;

FIG. 10 is an effect of Example 60 on EGF receptor autophosphorylationin A431 human epidermoid carcinoma;

FIG. 11 is an effect of Example 61 on EGF receptor autophosphorylationin A431 human epidermoid carcinoma;

FIG. 12 is an effect of Example 70 on EGF receptor autophosphorylationin A431 human epidermoid carcinoma;

FIG. 13 is a chart showing an inhibition of EGF receptor tyrosine kinaseby Example 27;

FIG. 14 is a graph showing an effect of Example 40 on growthfactor-mediated mitogenesis in Swiss 3T3 murine fibroblasts;

FIG. 15 is a photograph of an NIH 3T3 mouse fibroblast line, transfectedwith the human EGFR gene showing a normal flattened morphology;

FIG. 16 is a photograph of the same cell line treated with 100 ng/mL ofEGF showing a typical spindly transformed morphology; and

FIG. 17 is a photograph of the same cell line in the presence of both100 ng/mL of EGF and 5 μm of Example 27 showing the morphology revertedfrom the transformed type back to the normal type.

DESCRIPTION OF PREFERRED EMBODIMENTS

1. A preferred form of the invention has X=NH, n=0 or 1, in which caseR¹ =H, the aromatic ring phenyl optionally substituted, B, D & E carbon,with A nitrogen and R³ or R⁴ H, with the other one lower alkoxy orhalogen.

2. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one amino.

3. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one lower mono ordialkylamino.

4. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one hydrazino.

5. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one lower alkyl.

6. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ and R⁴ lower alkoxy.

7. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ and R⁴ lower alkyl.

8. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen, and R³ or R⁴ amino, with the other one loweralkoxy.

9. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen, and R³ or R⁴ lower mono or dialkylamino, withthe other one lower alkoxy.

10. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ lower mono or dialkylamino, with R⁴hydroxy.

A suitable ring structure for groups 1-10 is: ##STR3##

11. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen, and R³ and R⁴ taken together aredioxymethylene, dioxyethylene, 2,3-fused piperazine, 2,3-fusedmorpholine or 2,3-fused thiomorpholine. Suitable ring structures are:##STR4##

12. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, D & Ecarbon, with B nitrogen and R⁴ lower alkoxy or halogen.

13. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, D & Ecarbon, with B nitrogen and R⁴ amino.

14. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, D & Ecarbon, with B nitrogen and R⁴ lower mono or dialkylamino.

15. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, D & Ecarbon, with B nitrogen and R⁴ hydrazino.

16. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, C & Ecarbon, with B nitrogen and R⁴ lower alkyl.

A suitable ring structure for groups 12-16 is: ##STR5##

17. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ lower alkoxy or halogen.

18. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ amino.

19. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ lower mono or dialkylamino.

20. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ hydrazino.

21. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ amino.

A suitable ring structure for groups 17-21 is: ##STR6##

22. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one lower alkoxy.

23. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one amino.

24. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one lower mono ordialkylamino.

25. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one hydrazino.

26. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one lower alkyl.

27. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ and R⁴ lower alkoxy.

28. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ and R⁴ lower alkyl.

29. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ and R⁴ amino, with the other one loweralkoxy.

30. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ and R⁴ lower mono or dialkylamino, withthe other one lower alkoxy.

31. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ and R⁴ lower mono or dialkylamino, withR³ hydroxy.

A suitable ring structure for groups 22-31 is: ##STR7##

32. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen, and R³ and R⁴ taken together aredioxymethylene, dioxyethylene, 2,3-fused piperazine, 2,3-fusedmorpholine or 2,3-fused thiomorpholine.

33. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, A & D carbon, with B and E nitrogenand R⁴ lower alkoxy.

34. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, A & D carbon, with B and E nitrogenand R⁴ lower mono or dialkylamino.

35. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, A & D carbon, with B and E nitrogenand R⁴ amino.

36. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, A & D carbon, with B and E nitrogenand R⁴ hydrazino.

A suitable ring structure for groups 33-36 is: ##STR8##

37. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, B & D carbon, with A and E nitrogenand R³ and R⁴ lower alkoxy.

38. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, B & D carbon, with A and E nitrogenand R³ and R⁴ lower mono or dialkylamino.

39. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, B & D carbon, with A and E nitrogenand R³ or R⁴ lower alkoxy, with the other lower mono or dialkylamino.

40. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, B & D carbon, with A and E nitrogenand R³ and R⁴ taken together are ethylenedioxy, 2,3-fused piperazine,2,3-fused morpholine or 2,3-fused thiomorpholine.

A suitable ring structure for groups 37-40 is: ##STR9##

41. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase, R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a sulfur atom, with D & E carbon, or A & Bare carbon with D and E taken together as a sulfur atom, with R⁴ or R³H, lower alkyl, lower alkoxy, amino, or lower mono or dialkyl amino.

42. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are an oxygen atom, with D & E carbon, or A & Bare carbon with D and E taken together as an oxygen atom, with R⁴ or R³H, lower alkyl, lower alkoxy, amino, or lower mono or dialkylamino.

43. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a nitrogen atom, with D & E carbon, or A & Bare carbon with D and E taken together as a nitrogen atom, with R⁴ or R³H, lower alkyl, lower alkoxy, amino, or lower mono or dialkylamino.

44. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a sulfur atom with D carbon and E nitrogen,or D and E taken together are a sulfur atom, and A is nitrogen and B iscarbon, with R^(3/4) H, lower alkyl, lower alkoxy, amino, or lower monoor dialkylamino.

45. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are an oxygen atom with D carbon and E nitrogen,or D and E taken together are an oxygen atom, and A is nitrogen and B iscarbon, with R^(3/4) H, lower alkyl, lower alkoxy, amino, or lower monoor dialkylamino.

46. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A and Btaken together are a nitrogen atom, and D is carbon and E is nitrogen,with R^(3/6) H, or lower alkyl, and R⁴ H, lower alkyl, lower alkoxy,amino, or lower mono or dialkylamino.

47. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are an oxygen atom with D nitrogen and E carbon,or A and B taken together are a carbon atom with D nitrogen and Eoxygen, with R^(3/6) H, lower alkyl, lower alkoxy, amino, or lower monoor dialkylamino.

48. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a sulfur atom with D nitrogen and E carbon,or A and B taken together are a carbon atom with D nitrogen and Esulfur, with R^(3/6) H, lower alkyl, lower alkoxy, amino, or lower monoor dialkylamino.

49. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a nitrogen atom with D nitrogen and E carbon,or A and B taken together are a carbon atom with D and E nitrogen atoms,with R^(3/6) H or lower alkyl if on nitrogen, or H, lower alkyl, loweralkoxy, amino, or lower mono or dialkylamino if on carbon.

Other suitable ring structures are: ##STR10##

The compounds of the present invention are prepared according to anumber of alternative reaction sequences.

Preparative Routes to Compounds of the Invention

Scheme 1--Route for Preferred Groups 1-5, R⁴ =H

Displacement of the 2-chloro of 2,6-dichloro-3-nitropyridine is carriedout by cuprous cyanide in NMP. Displacement of the second chlorine ofthis nitrile by fluoride at this step can be advantageous. This isfollowed by a mild reduction of the nitro group, under conditions wherethe halogen is not hydrogenolysed. Hydrolysis of the nitrile followed byorthoformate cyclization, and Vilsmeier-type chlorination will give thedihalopyridopyrimidine. Displacement of the more reactive 4-chlorinewith an appropriate amine is followed by displacement of the 6-halogenwith the appropriate nucleophile, ammonia, lower alkylamine, hydrazine,methoxide, to form the final products. (NMP is a solvent,N-methyl-2-pyrrolidone).

1. A preferred form of the invention has X=NH, n=0 or 1, in which caseR¹ =H, the aromatic ring phenyl optionally substituted, B. D & E carbon,with A nitrogen and R³ or R⁴ H, with the other one lower alkoxy orhalogen.

2. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one amino.

3. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one lower mono ordialkylamino.

4. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one hydrazino.

5. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one lower alkyl.

Scheme 2--Route to Preferred Groups 1-5, R³ =H

Displacement of chlorine from 2-chloro-3,5-dinitropyridine isaccomplished with CuCN in NMP. Reduction of the nitro groups to aminesis followed by hydrolysis of the nitrile to an amide. This is cyclizedto the pyrimidone with orthoformate, which is converted to the chlorideby POCl₃ or possibly turned into the thiomethyl derivative by treatmentwith phosphorus pentasulfide followed by MeI and a mild base.Displacement with the appropriate amine gives the desired 7-aminocompound. The amine functionality can be reductively alkylated oractivated by diazotisation of the amino group under acidic or basicconditions, followed by a reduction to the hydrazide, or conversion intoa lower alkyl ether, or to a halogen followed by a cuprate or Stillecoupling by methods familiar to those skilled in the art. Alternatively,the amine can be reductively aminated, or acylated and reduced to formthe alkylamino side chain.

Scheme 3--Route to Preferred Groups 6 and 8-10 where R⁴ =RO

The known metalation of 2,6-difluoropyridine is exploited twice. LDAtreatment followed by a borate/hydrogen peroxide introduces the3-hydroxy substituent. If the pyridine undergoes the 2nd metalation atthe 4 position, the alcohol can be protected as a TIPS (triisopropylsilyl) ether, which will force the second metalation to the 5-position.Alternative nitrations may be used, such as converting the lithiumintermediate to a stannane and treatment with tetranitromethane, or theuse of NO₂ BF₄ (nitronium tetrafluoroborate). The C₁ displacement may beeffected by cuprous cyanide or other sources of cyanide ion. Afternitrile hydrolysis and nitro group reduction, ethyl orthoformate may beused instead of formamide for the cyclization, and it may be that somecyclizations will require displacement of F by MeS prior to thereaction. The 4-position is activated by chlorination, and the sidechainamine is then introduced. The final displacement can be by alkoxide oramine nucleophiles to generate the various dialkoxy and amino-alkoxyspecies, and the appropriate use of R can allow the 7-hydroxyl group tobe unmasked at the end of the synthesis. (LDA means lithium diisopropylamide).

6. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ and R⁴ lower alkoxy.

8. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen, and R³ or R⁴ amino, with the other one loweralkoxy.

9. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen, and R³ or R⁴ lower mono or dialkylamino, withthe other one lower alkoxy.

10. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ lower mono or dialkylamino, with R⁴hydroxy.

Scheme 4--Route to Preferred Group 7

Use of the 6-alkylquinaldic acid followed by ionic bromination underforcing conditions gives an anhydride, which is opened with ammonia,recyclized to the imide, and then the Hoffman degradation occurs at theless active carbonyl. Cyclization and ring side chain addition in thenormal manner is followed by a Stille coupling to introduce the R¹ alkylgroup. At this step alkenyl or aryl substituents could also beintroduced using this coupling technology.

7. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ and R⁴ lower alkyl.

Scheme 5--Route to Preferred Groups 8, 9, R³ =OR

Dinitration of 2,6-dihydroxypyridine is followed by conversion to thevery reactive dichlorocompound. The dinitrodichloropyridine is singlydisplaced by cuprous cyanide in NMP, and then the compound is reducedunder mild conditions to the diamine. The nitrile is hydrolysed to theamide, which can then be cyclized to the pyridopyrimidone, which is4-chlorinated in the usual fashion. Displacement of the more reactivechlorine with the 4-sidechain is followed by displacement of the6-chlorine with alkoxid. For group 9, the amine should be alkylatedappropriately by methods familiar to one skilled in the art.

Scheme 6--Route to Preferred Group 11

Compounds of preferred group 11 are specialized cases of preferredgroups 6, 8, 9 and 10, where R³ and R⁴ are cyclized together. They canbe made using the same routes as those described for the preferredgroups, with minor modifications, which will be obvious to one skilledin the art. For example vicinally substituted alkoxy amino compounds canbe dealkylated, and the corresponding vicinal aminoalcohols can bebisalkylated with an appropriate dihaloalkane.

11. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen, and R³ and R⁴ taken together aredioxymethylene, dioxyethylene, 2,3-fused piperazine, 2,3-fusedmorpholine or 2,3-fused thiomorpholine.

Scheme 7--Route for Preferred Groups 12-16

2,4-Diamino-5-cyanopyridine can be cyclized directly to many4-benzylamipnopyridopyrimidine derivatives by treatment with thebenzylamine and formic acid at high temperature. For less nucleophilicamines 2,4-diamino-5-cyanopyridine is converted via ethylorthoformate/acetic anhydride treatment, followed by cyclization withhydrosulfide ion in anhydrous conditions, to give7-amino-4-thiono-3H-pyrido[4,3-d]pyrimidine. S-Alkylation anddisplacement with an appropriate amine gives the desired product. If R⁴is not amino, the amine can be acylated, or reductively alkylated.Alternatively 2,4-diamino-5-cyanopyridine can be hydrolysed to thecorresponding amide, and this species can be cyclized to7-amino-4-oxo-3H-pyrido[4,3-d]pyrimidine with orthoformate.Diazotization of the 7-amine and replacement with fluorine allows forintroduction of other amine and alkoxide nucleophiles at the end of thesynthesis after the C4 substituent has been introduced in the usualmanner. Diazotization and replacement of the amine with bromide allowsfor Stille couplings at the 7-position.

12. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, D & Ecarbon, with B nitrogen and R⁴ lower alkoxy or halogen.

13. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, D & Ecarbon, with B nitrogen and R⁴ amino.

14. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, D & Ecarbon, with B nitrogen and R⁴ lower mono or dialkylamino.

15. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, D & Ecarbon, with B nitrogen and R⁴ hydrazino.

16. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, C & Ecarbon, with B nitrogen and R⁴ lower alkyl.

Scheme 8--Route for Preferred Groups 17-21

2-Chloro-5-nitropyridine is converted to the corresponding2-fluorocompound by KF in DMSO. Reduction of the nitro group followed bytreatment with Boc anhydride gives the Bocamino derivative, which can bemetalated and carboxylated at the 4-position. Removal of the Boc withTFA and cyclization of the pyrimidone ring with formamide gives6-fluoro-4-oxo-3H-pyrido[3,4-d]pyrimidine. This is 4-chlorinated in theusual manner and the 4-sidechain is introduced via displacement with anappropriate amine. Displacement of the 6-fluorine with appropriatenucleophiles leads to various different final products. If the fluorineis displaced by thiomethoxide, that in turn can be displaced by alkylgroups in Ni-catalyzed Grignard displacements.

17. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ lower alkoxy or halogen.

18. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ amino.

19. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ lower mono or dialkylamino.

20. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ hydrazino.

21. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Ecarbon, with D nitrogen and R³ lower alkyl.

Scheme 9--Route to Preferred Groups 22-26, R⁴ =H

Nitration of 2-methoxynicotinic acid is followed by displacement of theactivated methoxy group and cyclization of the pyrimidone ring, possiblyall in one step with formamidine, or alternatively in two steps withammonia followed by cyclization with a formamide equivalent. Thecarbonyl is converted to the chloride and displaced with the sidechainin the usual fashion, and the nitro group is then selectively reduced toamino. This can be alkylated, acylated or diazotized. The diazo compoundcan be converted to hydroxy or to the bromide or iodide, and theselatter can undergo a Stille coupling to introduce lower alkyl, alkenyl,aryl, etc. at R³.

22. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one lower alkoxy.

23. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one amino.

24. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one lower mono ordialkylamino.

25. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one hydrazino.

26. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ or R⁴ H, with the other one lower alkyl.

Scheme 10--Route to Preferred Groups 22-26, R³ =H

This route uses the known metalation and carboxylation of2,6-difluoropyridine, followed by displacement of the 2-fluorosubstituent. Cyclization of the pyrimidone ring with formamide, followedby conversion of the carbonyl into chloride in a normal manner gives achlorofluoropyridopyrimidine. The ar(alk)ylamino sidechain is introducedby displacement of the more reactive pyrimidine chlorine, and the R⁴substituent is then introduced by fluoride displacement. Theintroduction of alkyl utilizes displacement of by alkoxide, later ethercleavage to the pyridone, O-triflation and Stille coupling.

Scheme 11--Route to Preferred Groups 27 and 29-31, R³ =RO

This scheme relies on the metalation of 2,6-difluoropyridine similarlyto scheme 10. The first metalation is used to introduce oxygen, and thesecond to introduce the carboxylic acid. If required to force the secondmetalation to the 5-position the oxygen may be protected as the verybulky TIPS ether, and stronger bases than LDA may be required. Ammoniais introduced at the 2-position under high temperature and pressure, andthe pyridone ring is cyclized, and activated at the 4-position in theusual manner and then displaced with the 4-position sidechain.Displacement of the 7-fluoro substituent with an appropriatenucleophile, followed by conversions as described in previous schemesfinishes the synthesis.

27. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ and R⁴ lower alkoxy.

29. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen, and R³ or R⁴ amino, with the other one loweralkoxy.

30. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen, and R³ or R⁴ lower mono or dialkylamino, withthe other one lower alkoxy.

31. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R⁴ lower mono or dialkylamino, with R³hydroxy.

Scheme 12--Route to Preferred Group 28

5-Bromo-2,6-difluoronicotinic acid is prepared from 2,6-difluoropyridineby successive lithiations using LDA. The 5-position is alkylated via aStille coupling, and the pyrimidone ring is cyclized on in two steps.The 4-substituent is introduced in the usual fashion and the 7-fluorogroup is displaced with thiomethoxide. This thioether in turn isdisplaced by a Grignard agent in the presence of a nickel salt catalyst.Again use of appropriate organometallic reagents in the Stille andGrignard couplings could lead to alkenyl, alkynyl and aryl substituentsat R³ and R⁴.

28. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen and R³ and R⁴ lower alkyl.

Scheme 13--Route to Preferred Groups 29 and 30, R⁴ =RO

Nitration of the commercially available dichloronicotinic acid isfollowed by a selective displacement of the more reactive Cl under mildconditions, followed by a more forcing displacement of the other Cl, inth6-appropriate order. The resulting 6-alkoxy-2-amino-5-nitronicotinicacid is cyclized to the pyrimidone, and the 4-carbonyl is converted to achloride and displaced in the usual fashion with an appropriate amine togive the 4-amino-7-alkoxy-6-nitropyrido[2,3-d]pyrimidine. Reduction ofthe nitro group, followed by any desired alkylation or acylation givesthe desired compounds.

Scheme 14--Route to Preferred Group 32

Compounds of group 32 are specialized cases of preferred groups 27, 29,30 and 31, where R³ and R⁴ are cyclized together. They can be made usingthe same routes as those described for these preferred groups with minormodifications. For example, vicinally substituted alkoxy amino compoundscan be dealkylated, and the corresponding vicinal amino alcohols can bebisalkylated with an appropriate dihaloalkane. Piperazines can be madeby the route shown in Scheme 13, provided that a suitable aminenucleophile is used to displace the 6-chloro substituent instead of analkoxide.

32. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A, B & Dcarbon, with E nitrogen, and R³ and R⁴ taken together aredioxymethylene, dioxyethylene, 2,3-fused piperazine, 2,3-fusedmorpholine or 2,3-fused thiomorpholine.

Scheme 15--Route to Preferred Groups 33-36

Reaction of a suitable S-alkylisothiouronium salt withmethoxymethylidine malononitrile yields a fully functionalized pyrimideprecursor. The initially formed pyrimidine can have the SEt displaced byR⁴ either before or after the nitrile hydrolysis, if displacement oroxidation prove problematic later. Displacement of the SEt group canalso be achieved without an oxidation to activate the sulfur.Cyclization of the second pyrimidine ring is followed by activation ofthe 4-carbonyl by thiation and alkylation. Even if the 7-thio group hasnot been displaced at this point, introduction of the 4-amino sidechainoccurs preferentially.

33. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, A & D carbon, with B and E nitrogenand R⁴ lower alkoxy.

34. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, A & D carbon, with B and E nitrogenand R⁴ lower mono or dialkylamino.

35. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, A & D carbon, with B and E nitrogenand R⁴ amino.

36. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, A & D carbon, with B and E nitrogenand R⁴ hydrazino.

Scheme 16--Route to Preferred Groups 37-40

The pterine nucleus is made by well-established procedure. For group 37,the pterindione intermediate can be O-alkylated, and for it, and theother groups, the pterindione can be converted to the trichloropterin,and selective displacements can be carried out on the halogens in anorder appropriate to give the desired compound.

37. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, B & D carbon, with A and E nitrogenand R³ and R⁴ lower alkoxy.

38. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, B & D carbon, with A and E nitrogenand R³ and R⁴ lower mono or dialkylamino.

39. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, B & D carbon, with A and E nitrogenand R³ or R⁴ lower alkoxy, with the other lower mono or dialkylamino.

40. Another preferred form of the invention has X=NH, n=0, the aromaticring phenyl optionally substituted, B & D carbon, with A and E nitrogenand R³ and R⁴ taken together are ethylenedioxy, 2,3-fused piperazine,2,3-fused morpholine or 2,3-fused thiomorpholine.

Scheme 17--Route to Preferred Groups 41 [3,2-d] Ring Fusion

3,H-Thieno[3,2-d]pyrimid-4-one can be made by standard chemistry fromcommercially available ethyl 3-aminothiophene carboxylate and formamide.Conversion of the carbonyl to chloride by standard techniques followedby displacement with an appropriate amine gives the desiredthieno[3,2-d]pyrimidines. If R⁴ is not H, an appropriate electrophile,for example nitro for amine based or diazotization derived substituents,or Br for Stille coupled final products, can be introduced either at thestage shown or an earlier stage, and then be converted to R⁴, byreduction and amination for example or by Stille coupling, or othermethods known to those skilled in the art. [This technique follows alsofor all of the following preferred categories which have the possibilityof substitution on R³ or R⁴, as they are all contain electron rich fivemembered rings which can be readily manipulated by electrophilicaromatic substitution.] (DMSO is dimethyl sulfoxide).

41. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a sulfur atom, with D & E carbon, or A & Bare carbon with D and E taken together as a sulfur atom, with R⁴ or R³H, lower alkyl, lower alkoxy, amino, or lower mono or dialkylamino.

Scheme 18--Route to Preferred Groups 41 [2,3-d] Ring Fusion

Thieno[2,3-d]pyrimid-4-one is built up by the Gewald synthesis from2,5-dihydroxy-1,4-dithiane and ethyl cyanoacetate, followed by formamidecyclization. Conversion of the carbonyl to chloride by standardtechniques followed by displacement with an appropriate amine gives thedesired thieno[2,3-d]pyrimidines.

Scheme 19--Route to Preferred Groups 42 [3,2-d] Ring Fusion

The [3,2-d] ring fusion compounds are obtained from 3-bromofurfural asshown above in Scheme A. Displacement of the bromide by azide, followedby is oxidation of the aldehyde sets up the basic aminofuroic acidneeded to fuse on the pyrimidine ring. The annulation shown can be used,or by manipulating which acid derivative is actually used, one could usea variety of other ring annulations, and subsequent activations of the4-position if required.

42. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are an oxygen atom, with D & E carbon, or A & Bare carbon with D and E taken together as an oxygen atom, with R⁴ or R³H, lower alkyl, lower alkoxy, amino, or lower mono or dialkylamino.

Scheme 20--Route to Preferred Groups 42 [2,3-d] Ring Fusion

Reaction of 6-chloro-4-methylthiopyrimidine with LDA followed by DMFgives the corresponding 5-aldehyde, which is treated with the sodiumsalt of an appropriate glycollate ester, displacing chlorine, and insitu forming the furan ring by intramolecular aldol condensation.Cleavage of the ester and decarboxylation of the unwanted 7-acidfunctionality may be done in a single reaction with a good nucleophilein a dipolar aprotic solvent at high temperature, or in separatesaponification and Cu/quinoline decarboxylation steps. Displacement ofthe 4-methylthio group by an appropriate amine gives the desiredfurano[2,3-d]pyrimidines.

Scheme 21--Route to Preferred Groups 43 [2,3-d] Ring Fusion

To make the pyrrolo[2,3-d]pyrimidine a pyrimidine ring is cyclized ontothe cyano amino-pyrrole using known techniques as shown in scheme Babove. Activation and displacement of the thiol by the side chain can bepreceded or followed by the optional electrophilic substitution of thepyrrole ring.

43. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a nitrogen atom, with D & E carbon, or A & Bare carbon with D and taken together as a nitrogen atom, with R⁴ or R³ Hlower alkyl, lower alkoxy, amino, or lower mono or dialkylamino.

Scheme 22--Route to Preferred Groups 43 [3,2-d] Ring Fusion

The preparation of the pyrrolo[3,2-d]pyrimidine exploits the knowncondensation of orthoformate with the acidified 4-methyl group of6-pyrimidones to form the pyrrolopyrimidine as shown above. The sidechain can be put on by standard techniques such as in Scheme 1, and theR⁴ substituent can be introduced by standard electrophilic chemistry asdescribed above.

Scheme 23--Route to Preferred Groups 44 [5,4-d] Ring Fusion

Condensation of dithioformic acid with 2-aminomalononitrile in thepresence of a dehydrating agent such as PPA gives5-amino-4-cyanothiazole. Reaction of this with orthoformate, followed bytreatment with MeSNa gives a thiazolo[5,4-d]pyrimidine derivative, whichon treatment with an appropriate amine give the desired compounds.

44. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a sulfur atom with D carbon and E nitrogen,or D and E taken together are a sulfur atom, and A is nitrogen and B iscarbon, with R^(3/4) H, lower alkyl, lower alkoxy, amino, or lower monoor dialkylamino.

Scheme 24--Route to Preferred Groups 44 [4,5-d] Ring Fusion

Reaction of N-cyanobismethylthiomethyl-eneimine with ethylthioglycollate gives ethyl 2-methylthio-4-aminothiazole-5-carboxamide.Cyclization with formamide or equivalent, followed by desulfurization ofthe methylthio gives a thiazolopyrimidone, which can be activated byVilsmeier reagent, and the chloride displaced by the desired amine togive the desired thiazolo[4,5-d]pyrimidine derivatives as shown above.

Scheme 25--Route to Preferred Groups 45 [5,4-d] Ring Fusion

The known 5-amino-4-cyanooxazole is treated with ethylorthoformate/acetic anhydride, and is then reacted with MeSNa to give4-methylthiooxazolo[5,4-d]pyrimidine, which on displacement with theappropriate amine gives the desired oxazolo[5,4-d]pyrimidines as shownabove.

45. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are an oxygen atom with D carbon and E nitrogen,or D and E taken together are an oxygen atom, and A is nitrogen and B iscarbon, with R^(3/4) H, lower alkyl, lower alkoxy, amino, or lower monoor dialkylamino.

Scheme 26--Route to Preferred Groups 45 [4,5-d] Ring Fusion

Diazotization of the known 5-amino-4,6-dichloropyrimidine, followed bydilute sulfuric acid treatment give the corresponding 5-hydroxycompound. One of the chlorines is displaced with ammonia, and theoxazole ring is annulated with formic acid or an appropriate equivalent.Displacement of the other chlorine with an appropriate amine gives thedesired oxazolo[4,5-d]pyrimidines as shown above

Scheme 27--Route to Preferred Groups 46

These compounds can be made by straightforward displacement of halogenon appropriate 6-chloropurines, by means well documented in the art. R³substituents can be introduced via facile electrophilic substitutions atthe activated 8-position of the purine nucleus, followed by the types oftransformation discussed in previous examples.

46. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, A and Btaken together are a nitrogen atom, and D is carbon and E is nitrogen,with R^(3/6) H, or lower alkyl, and R⁴ H, lower alkyl, lower alkoxy,amino, or lower mono or dialkylamino.

Scheme 28--Route to Preferred Groups 47 [5,4-d] Ring Fusion

Reaction of 6-chloro-4-methylthiopyrimidine with LDA followed by DMFgives the corresponding 5-aldehyde, which is treated with hydroxylamineunder mild acidic conditions, and then basic conditions to complete thering annulation giving 4-methylthioisoxazolo[5,4-d]pyrimidine, which ondisplacement with an appropriate amine gives the desiredisoxazolo[5,4-d]pyrimidine derivatives as shown above.

47. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are an oxygen atom with D nitrogen and E carbon,or A and B taken together are a carbon atom with D nitrogen and Eoxygen, with R^(3/6) H, lower alkyl, lower alkoxy, amino, or lower monoor dialkylamino.

Scheme 29--Route to Preferred Groups 47 [4,5-d] Ring Fusion

Reaction of 4,6-dichloro-5-nitropyrimidine with CuCN/NMP gives the4-nitrile. Reduction of the nitro group to the corresponding amine isfollowed by diazotization and treatment with dilute sulfuric acid togive the corresponding 5-hydroxy compound. Reaction of this with Me₃Al/NH₄ Cl gives the amidine which is oxidatively cyclized to7-amino-4-chloroisoxazolo[4,5-d]pyrimidine. Removal of the aminofunctionality by diazotization/hypophosphorus acid is followed bydisplacement of the 4-chlorine with an appropriate amine to give thedesired isoxazolo[4,5-d]pyrimidine derivatives as shown above.

Scheme 30--Route to Preferred Groups 48 [5,4-d] Ring Fusion

Reaction of 6-chloro-4-methylthiopyrimidine with LDA followed by DMFgives the corresponding 5-aldehyde, which is treated sequentially withNaSH, NBS and ammonia to complete the ring annulation giving4-methylthioisothiazolo[5,4-d]pyrimidine, which on displacement with anappropriate amine gives the desired isothiazolo[5,4-d]pyrimidinederivatives as shown above.

48. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a sulfur atom with D nitrogen and E carbon,or A and B taken together are a carbon atom with D nitrogen and Esulfur, with R^(3/6) H, lower alkyl, lower alkoxy, amino, or lower monoor dialkylamino.

Scheme 31--Route to Preferred Groups 48 [4,5-d] Ring Fusion

Reaction of 4,6-dichloro-5-nitropyrimidine with CuCN/NMP gives the4-nitrile. Reduction of the nitro group to the amine is followed bydiazotization/thiation to give the corresponding 5-mercapto compound.Reaction of this with Me₃ Al/NH₄ Cl gives the amidine which isoxidatively cyclized with NBS to7-amino-4-chloroisothiazolo[4,5-d]pyrimidine. Removal of the aminofunctionality by diazotization/hypophosphorus acid is followed bydisplacement of the 4-chlorine with an appropriate amine to give thedesired isothiazolo[4,3-d]pyrimidine derivatives as shown above.

Scheme 32--Route to Preferred Groups 49 [3,4-d] Ring Fusion

Reaction of 6-chloro-4-methylthiopyrimidine with LDA followed by DMFgives the corresponding 5-aldehyde, which is treated with hydrazine todo the ring annulation giving 4-methylthiopyrazolo[3,4-d]pyrimidine,which on displacement with an appropriate amine gives the desiredpyrazolo[3,4-d]pyrimidine derivatives as shown above.

49. Another preferred form of the invention has X=NH, n=0 or 1, in whichcase R¹ =H, the aromatic ring phenyl optionally substituted, and eitherA and B taken together are a nitrogen atom with D nitrogen and E carbon,or A and B taken together are a carbon atom with D and E nitrogen atoms,with R^(3/6) H or lower alkyl if on nitrogen, or H, lower alkyl, loweralkoxy, amino, or lower mono or dialkylamino if on carbon.

Scheme 33--Route to Preferred Groups 49 [4,3-d] Ring Fusion

Nitration of pyrazole-3-carboxylic acid followed by reduction gives4-aminopyrazole-3-carboxylic acid. This is cyclized topyrazolo[4,3-d]pyrimid-4-one with formamidine HCl, and replacement ofthe carbonyl with halide by standard procedures, followed bydisplacement of the chloride by an appropriate amine yields the desiredpyrazolo[4,3-d]pyrimidine, as shown above.

Most Preferred Forms of the Invention

1. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, B, D & E are carbon, A is nitrogen, andR⁴ is amino.

2. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, B, D & E are carbon, A is nitrogen, andR⁴ is methylamino.

3. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, B, D & E are carbon, A is nitrogen, andR⁴ is dimethylamino.

4. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-nitrophenyl, A, D & E are carbon, B is nitrogen, andR⁴ is amino.

5. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, B is nitrogen, andR⁴ is amino.

6. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 4-bromophenyl, A, D & E are carbon, B is nitrogen, andR⁴ is amino.

7. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-trifluoromethylphenyl, A, D & E are carbon, B isnitrogen, and R⁴ is amino.

8. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E ate carbon, B is nitrogen, andR⁴ is acetylamino.

9. A most preferred form of the invention is one where X=NH, x=1, R¹ =H,the aromatic ring is phenyl, A, D & E are carbon, B is nitrogen.

10. A most preferred form of the invention is one where X=NH, x=1, R¹=H, the aromatic ring is phenyl, A, D & E are carbon, B is nitrogen, andR⁴ is acetylamino.

11. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, B & E are carbon, D is nitrogen, R³Cl.

12. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, D is nitrogen, andR³ is methoxy.

13. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, D is nitrogen, andR³ is methylamino.

14. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, D is nitrogen, andR³ is dimethylamino.

15. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, D & E are carbon, and A and B takentogether are S.

16. A most preferred form of the invention is one where X=NH, x=1, R¹=H, the aromatic ring is phenyl, D & E are carbon, and A and B takentogether are S.

17. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A & B are carbon, and D and E takentogether are S.

18. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, B is carbon, and A, and D and E takentogether, are nitrogen.

19. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, B & E are carbon, D is nitrogen, andR³ is N-piperinyl.

20. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, B is nitrogen, andR⁴ is fluoro.

21. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-hydroxyphenyl, A, D & E are carbon, B is nitrogen,and R⁴ is amino.

22. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, B is nitrogen, andR⁴ is methylamino.

23. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, B is nitrogen, andR⁴ is dimethylamino.

24. A most preferred form of the invention is one where X=NMe, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, B is nitrogen, andR⁴ is methylamino.

25. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, D & E are carbon, B is nitrogen, andR⁴ is methoxy.

26. A most preferred form of the invention is one where X=NH, x=0, thearomatic ring is 3-bromophenyl, A, B & D are carbon, E is nitrogen, andR⁴ is fluoro.

Biology

These compounds are potent and selective inhibitors of the human EGFreceptor tyrosine kinase and other members of the EGF receptor family,including the ERB-B2, ERB-B3 and ERB-B4 receptor kinases, and are usefulfor the treatment of proliferative diseases in mammals. These inhibitorsprevent mitogenesis in cells where mitogenesis is driven by one or moreof this family of receptor kinases. This can include normal cells, whereit is desired to prevent mitogenesis, as exemplified by the cellstransformed by overexpression or mutation of this kinase family asexemplified by poor prognosis breast cancer where overexpression ofEGFR, ERB-B2 and ERB-B3 or mutation of ERB-B2 to the oncoprotein NEU isa major factor in cellular transformation. As the preferred compoundsare not highly cytotoxic and do not show potent growth inhibitoryproperties, because of their high specificity toward inhibition of theEGFR kinase family, they should have a much cleaner toxicity profilethan most anti-cancer and anti-proliferative drugs. Their very differentmode of action to current anti-cancer drugs should allow for their usein multiple drug therapies, where synergism with available agents isanticipated.

Compounds of the invention have been shown to be very potent, reversibleinhibitors of the EGF receptor tyrosine kinase, by binding with highaffinity at the adenosine triphosphate (ATP) binding site of the kinase.These compounds exhibit potent IC₅₀ s, varying from 10 micromolar to 5picomolar, for the tyrosine kinase activity of the enzyme, based on anassay examining phosphorylation of a peptide derived from thephosphorylation site of the protein PLCgamma1, a known EGFRphosphorylation substrate. This data is shown in Table 1.

Biological Data

Materials and Methods

Purification of Epidermal Growth Factor Receptor Tyrosine Kinase--HumanEGF receptor tyrosine kinase was isolated from A431 human epidermoidcarcinoma cells which overexpress EGF receptor by the following methods.Cells were grown in roller bottles in 50% Delbuco's Modified Eagle and50% HAM F-12 nutrient media (Gibco) containing 10% fetal calf serum.Approximately 10⁹ cells were lysed in two volumes of buffer containing20 mM 2-(4N-[2-hydroxyethyl]piperazin-1-yl)ethanesulfonic acid (hepes),pH 7.4, 5 mM ethylene glycol bis(2-aminoethyl ether)N,N,N',N'-tetraacetic acid, 1% Triton X-100, 10% glycerol, 0.1 mM sodiumorthovanadate, 5 mM sodium fluoride, 4 mM pyrophosphate, 4 mM benzamide,1 mM dithiothreitol, 80 μg/mL aprotinin, 40 μg/mL leupeptin and 1 mMphenylmethylsulfonyl fluoride. After centrifugation at 25,000×g for 10minutes, the supernatant was equilibrated for 2 h at 4° C. with 10 mL ofwheat germ agglutinin sepharose that was previously equilibrated with 50mM Hepes, 10% glycerol, 0.1% Triton X-100 and 150 mM NaCl, pH 7.5,(equilibration buffer). Contaminating proteins were washed from theresin with 1M NaCl in equilibration buffer, and the enzyme was elutedwith 0.5M N-acetyl-1-D-glucosamine in equilibration buffer, followed by1 mM urea. The enzyme was eluted with 0.1 mg/ml EGF. The receptorappeared to be homogeneous as assessed by Coomassie blue stainedpolyacrylamide electrophoretic gels.

Determination of IC₅₀ values--enzyme assays for IC₅₀ determinations wereperformed in a total volume of 0.1 mL, containing 25 mM Hepes, pH 7.4, 5mM MgCl₂, 2 mM MnCl₂, 50 μM sodium vanadate, 5-10 ng of EGF receptortyrosine kinase, 200 μM of a substrate peptide,(Ac-Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr⁴⁷² -Glu-Glu-Val-NH₂ ;derived from the amino acid (Tyr⁴⁷² has been shown to be one of fourtyrosines in PLC (phospholipaseC)-gamma 1 that are phosphorylated by theEGF receptor tyrosine kinase [Wahl, M. I.; Nishibe, S.; Kim, J. W.; Kim,H.; Rhee, S. G.; Carpenter, G., J. Biol. Chem., (1990), 265,3944-3948.], and peptides derived from the enzyme sequence surroundingthis site are excellent substrates for the enzyme.), 10 μM ATPcontaining 1 μCi of [³² P]ATP and incubated for ten minutes at roomtemperature. The reaction was terminated by the addition of 2 mL of 75mM phosphoric acid and passed through a 2.5 cm phosphocellulose filterdisc to bind the peptide. The filter was washed five times with 75 mMphosphoric acid and placed in a vial along with 5 mL of scintillationfluid (Ready gel Beckman).

                  TABLE 1                                                         ______________________________________                                        EGF Receptor Tyrosine Kinase Inhibition                                              Example #                                                                            IC.sub.50                                                       ______________________________________                                                1     8             μM                                                     2     3.6           μM                                                     3     1.1           μM                                                     4     225           nM                                                        5     1.9           μM                                                     6     7.6           nM                                                        7     3.1           nM                                                        8     9.6           nM                                                        9     405           nM                                                       10     6.1           μM                                                    11     194           nM                                                       12     13            nM                                                       13     250           nM                                                       14     70            nM                                                       15     134           nM                                                       16     3.7           μM                                                    17     1.55          μM                                                    18     173           nM                                                       19     1.8           μM                                                    20     4.9           μM                                                    21     1.25          μM                                                    22     39            nM                                                       23     840           nM                                                       24     123           nM                                                       25     377           nM                                                       26     241           nM                                                       27     10            nM                                                       28     94            nM                                                       29     262           nM                                                       30     10            μM                                                    31     15            nM                                                       32     4.7           μM                                                    33     130           pM                                                       34     91            pM                                                       35     3.1           nM                                                       36     29            nM                                                       37     39            nM                                                       38     71            nM                                                       39     590           nM                                                       40     578           nM                                                       41     220           nM                                                       42     226           nM                                                       43     10            μM                                                    44     10            μM                                                    45     2.87          μM                                                    46     1.42          μM                                                    47     1.67          μM                                                    48     1.0           μM                                                    49     2.5           μM                                                    50     10            μM                                                    51     1.95          μM                                                    52     8             μM                                                    53     1.8           μM                                                    54     100           nM                                                       55     400           nM                                                       56     110           nM                                                       57     124           nM                                                       58     40            nM                                                       59     2.6           nM                                                       60     8             pM                                                       61     6             pM                                                       62     6.1           μM                                                    63     6.1           μM                                                    64     11            nM                                                       65     5.1           μM                                                    66     190           nM                                                       67     6.1           μM                                                    68     263           nM                                                       69     7.0           μM                                                    70     473           nM                                                       71     11            nM                                                       72     35            nM                                                       73     36            nM                                                       74     11.5          μM                                                    75     55            nM                                                       76     10            μM                                                    77     3.9           nM                                                       78     670           nM                                                       79     6.7           nM                                                ______________________________________                                    

Cells

Swiss 3T3 mouse fibroblasts, A431 human epidermoid carcinoma cells, andMCF-7 (Michigan Cancer Foundation human mammary carcinoma cells),SK-BR-3 (human mamary carcinoma cells), MDA-MB-231 and MDA-MB-468 (humanmammary carcinoma cells) breast carcinomas were obtained from theAmerican Type Culture Collection, Rockville, Md. and maintained asmonolayers in dMEM (Dulbecco's modified eagle medium)/F12, 50:50(Gibco/BRL) containing 10% fetal bovine serum. To obtain conditionedmedium, MDA-MB-231 cells were grown to confluency in an 850 cm² rollerbottle and the medium-replaced with 50 ml of serum-free medium. After 3days the conditioned medium was removed, frozen down in aliquots andused as a heregulin source to stimulate erbB-2, 3, 4.

Antibodies

Monoclonal antibodies raised to the PDGF (platelet-desired growthfactor) receptor or phosphotyrosine were from Upstate Biotechnology,Inc., Lake Placid, N.Y. Anti-pp39^(jun) (antibody to the transcriptionfactor c-jun, which is a 39 kDalton phosphoprotein) and anti-EGFreceptor antibodies were from Oncogene Science, Uniondale, N.Y.

Immunoprecipitation and Western Blot

Cells were grown to 100% confluency in 100 mm Petrie dishes (Corning).After the cells were treated for 5 minutes with either EGF (epidermalgrowth factor), PDGF, or bFGF (basic fibroblast growth factor) (20ng/ml) or 1 ml of conditioned media from MDA-MB-231 cells, the media wasremoved and the monolayer scraped into 1 ml of ice cold lysis buffer (50mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% triton X-100, 1 mM EDTA,1 mM EGTA, 10 mM sodium pyrophosphate, 30 mM p-nitrophenyl phosphate, 1mM orthovanadate, 50 mM sodium fluoride, 1 mMphenylmethylsulfonylfluoride, 10 μg/ml of aprotinin, and 10 μg/ml ofleupeptin). The lysate was transferred to a microfuge tube (smallcentrifuge that holds 1-2 ml plastic centrifuge tubes), allowed to siton ice 15 minutes and centrifuged 5 minutes at 10,000×g. The supernatantwas transferred to a clean microfuge tube and 5 μg of antibody was addedto designated samples. The tubes were rotated for 2 hours at 4° C. afterwhich 25 μl of protein A sepharose was added and then rotation continuedfor at least 2 more hours. The protein A separose was washed 5 timeswith 50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol and 0.02% sodiumazide. The precipitates were resuspended with 30 μl of Laemlli buffer(Laemmli, NATURE, Vol. 727, pp. 680-685, 1970), heated to 100° C. for 5minutes and centrifuged to obtain the supernatant. Whole cell extractswere made by scraping cells grown in the wells of 6 well plates into 0.2ml of boiling Laemmli buffer. The extract were transferred to amicrofuge tube and heated to 100° C. for 5 minutes. The entiresupernatant from the immunoprecipitation or 35 μl of the whole cellextract was loaded onto a polyacrylamide gel (4-20%) and electrophoresiscarried out by the method of Laemlli (Laemmli, 1970). Proteins in thegel were electrophoretically transferred to nitrocellulose and themembrane was washed once in 10 mM Tris buffer, pH 7.2, 150 mM NaCl,0.01% Azide (TNA) and blocked overnight in TNA containing 5% bovineserum albumin and 1% ovalbumin (blocking buffer). The membrane wasblotted for 2 hours with the primary antibody (1 μg/ml in blockingbuffer) and then washed 2 times sequentially in TNA, TNA containing0.05% Tween-20 and 0.05% Nonidet P-40 (commercially available detergent)and TNA. The membranes were then incubated for 2 hours in blockingbuffer containing 0.1 μCi/ml of [¹²⁵ I] protein A and then washed againas above. After the blots were dry they were loaded into a film cassetteand exposed to X-AR X-ray film for 1-7 days. Protein A is a bacterialprotein that specifically bonds certain IgG subtypes and is useful inbinding to and isolating antibody-antigen complexes.

Northern Blots

Total cellular RNA was isolated from untreated control or treated Swiss3T3 cells using RNAzol-B (trademark of Tel Test Inc. for a kit used toisolate RNA from tissues) and adhered to the protocol described by themanufacturer. Forty to fifty μg of RNA was loaded onto a 1% agarose geland electrophoresis carried out for 3-4 hours at 65 volts. The RNA inthe gel was transferred by capillary action to a nylon membrane(Hybond-N, Amersham). The 40 mer c-jun probe was end labeled with [³²P]ATP using T4 nucleotide kinase (Promega) and purified on a G25sephadex column according to the procedure recommended by the supplier,Oncogene Science. Hybridization was performed overnight at 65° C. (c-junis an immediate early transcription factor; it is one of the componentsof AP-1 while FOS is the second component of AP-1.

Growth Factor-Mediated Mitogenesis

Swiss 3T3 fibroblasts were grown to 90-100% confluency in 24-well plates(1.7×1.6 cm, flat bottom) and growth arrested in serum-free media for 18hours. Drug was added to specified wells 2 hours prior to growth factorsand then the cells were exposed to either 20 ng/ml EGF, PDGF or bFGF or10% serum for 24 hours. Two μCi of [methyl-³ H]thymidine was added toeach well and incubated for 2 hours at 37° C. The cells were trypsinizedand injected into 2 ml of ice cold 15% trichloroacetic acid (TCA). Theresulting precipitate was collected on glassfiber filters, washed fivetimes with 2-ml aliquots of ice-cold 15% TCA, dried and placed inscintillation vials along with 10 ml Ready gel (Beckman, Irvine,Calif.). Radioactivity was determined in a Beckman LS 6800 scintillationcounter.

Growth Inhibition Assay

Cells (2×10⁴) were seeded in 24-well plates (1.7×1.6 cm, flat bottom) intwo mls of medium with or without various concentrations of drug. Plateswere incubated for 3 days at 37° in a humidified atmosphere containing5% CO₂ in air. Cell growth was determined by cell count with a CoulterModel AM electronic cell counter (Coulter Electronics, Inc., Hialeah,Fla.).

    ______________________________________                                        INHIBITION OF EGF-INDUCED AUTOPHOSPHORYLATION                                 IN A431 EPIDERMOID CARCINOMA CELLS AND CONDITIONED                            MEDIA-INDUCED AUTOPHOSPHORYLATION IN SK-BR-3 BREAST                           TUMOR CELLS BY COMPOUNDS OF THE CURRENT INVENTION                             Example #                                                                             EGFR IC.sub.50 nM                                                                         A431 IC.sub.50 nM                                                                         SKBR-3 IC.sub.50 nM                           ______________________________________                                         4      225         >1000       >10,000                                        6      7.6         53          2660                                           7      3.1         20          100                                            8      9.6         32          71                                            22      39          252         ˜1500                                   27      10          110         ˜800                                    59      2.6         12          <10                                           60      0.008       13          <10                                           61      0.006       21          39                                            70      11          124         <10                                           74      55          >1000       >1000                                         ______________________________________                                        ANTIPROLIFERATIVE PROPERTIES OF                                               TYROSINE KINASE INHIBITORS IC.sub.50 (nM)                                                     Ex 60  Ex 61                                                  ______________________________________                                        B104-1-1        2100   1000                                                   SK-BR-3         600    900                                                    MDA-468         3000   12000                                                  ______________________________________                                         B104-1-1-NIH-3T3 fibroblasts transfected by the neu oncogene, Stem et al.     Science, 234, pp. 321-324 (1987)                                              SKBR-3  Human breast carcinoma overexpressing erbB2 and erbB3                 MDA468  Human breast carcinoma overexpressing the EGF receptor           

The above gels, developed as detailed in the experimental section,demonstrate the efficacy of compounds of the current invention atblocking certain EGF-stimulated mitogenic signalling events in wholecells. The numbers to the left of the gels indicated the positions ofmolecular weight standards in kiloDaltons. The lane labelled controlshows the degree of expression of the growth-related signal in theabsence of EGF stimulation, whereas the lane labelled EGF (or PDGF orb-FGF) shows the magnitude of the growth factor-stimulated signal. Theother lanes show the effect of the stated quantities of the named drugon the growth factor-stimulated activity being measured, demonstratingthat the compounds of the present invention have potent effects in wholecells, consistent with their ability to inhibit the tyrosine kinaseactivity of the EGF receptor.

Gel of Example 40 (FIG. 7) detects mRNA for c-jun by hybridization witha specific radiolabelled RNA probe for c-jun. The gel demonstrates thatthe growth factors EFG, PDGF and b-FGF stimulate c-jun production inSwiss 3T3 cells, and that compound 40 blocks this production forEGF-stimulated cells, but not for PDGF or b-FGF stimulated cells.

Effect of Example 40 on Growth Factor Mediated Expression of p39^(c-jun)

This gel shows the amount of c-jun induced in Swiss 3T3 cells by thegrowth factor EGF, PDGF and b-FGF, quantitating with ananti-c-jun-specific monoclonal antibody. It demonstrates the ability ofExample 40 to block c-jun expression in Swiss 3T3 when stimulated byEGF, but not when stimulated by PDGF or b-FGF.

It is to be appreciated that the compounds described herein can be usedin combination with other components to enhance their activity. Suchadditional components are anti-neoplastic materials as, doxorubicin,taxol, cis platin, and the like.

It has been found that the compounds described herein may inhibit boththe erb-B2 and erb-B4 receptors and therefore have significantlyincreased clinical activity advantageously in combination with theaforementioned anti-neoplastic agents.

See also the results shown in FIGS. 1-17.

Some preferred structures are as follows:

    ______________________________________                                         ##STR11##                                                                    Ex #               Z                                                          ______________________________________                                        fluorine                                                                      6                  --NH.sub.2                                                 7                  --NHCH.sub.3                                               8                  --N(CH.sub.3).sub.2                                        ______________________________________                                         ##STR12##                                                                    Ex #            Z           R.sub.2                                           ______________________________________                                        22              NH.sub.2    --NO.sub.2                                        27              NH.sub.2    Br                                                ______________________________________                                         ##STR13##                                                                    Ex #           Z              R.sub.2                                         ______________________________________                                        59             --OCH.sub.3    Br                                              60             --NHCH.sub.3   Br                                              61             --N(CH.sub.3).sub.2                                                                          Br                                              ______________________________________                                    

Chemical Experimental

Listed below are preferred embodiments wherein all temperatures are indegrees Centigrade and all parts are parts by weight unless otherwiseindicated.

EXAMPLE 1 4-Anilinopyrido[3,2-d]pyrimidine mesylate

3H-Pyrido[3,2-d]pyrimidin-4-one. A solution of6-chloro-3-nitropicolinamide (2.00 g, 9.91 mmol) in EtOAc/MeOH (1:1, 100mL) is hydrogenated over 5% Pd-C (0.40 g) at 60 psi for 6 days, withadditions of fresh catalyst after 2 and 4 days. After removal of thecatalyst by filtration the solution is concentrated to dryness, to give3-aminopicolinamide as an orange oil, which is used directly in the nextstep. The crude product is stirred under reflux with triethylorthoformate (50 mL) for 42 h, during which time a tan precipitateforms. After cooling, the solid is filtered off, washed well withpetroleum ether, and dried under vacuum to give3H-pyrido[3,2-d]pyrimidin-4-one (1.27 g, 87%), mp 343-345° C. [Price, C.C. and Curtin, D. Y. J. Amer. Chem. Soc. 68, 914, 1946 report mp346-347° C.].

4-Chloropyrido[3,2-d]pyrimidine. A suspension of the above pyrimidinone(1.00 g, 6.80 mmol) in POCl₃ (30 mL) is heated under reflux for 4 h, andthen concentrated to dryness under reduced pressure. The residue ispartitioned between CH₂ Cl₂ and saturated NaHCO₃ solution, and theorganic layer worked up to give 4-chloropyrido[3,2-d]pyrimidine (0.97 g,86%) as a tan solid, mp 335° C. (dec), which is used without furthercharacterisation.

4-Anilinopyrido[3,2-d]pyrimidine mesylate. A solution of4-chloropyrido[3,2-d]pyrimidine (84 mg, 0.5 mmol), aniline (56 mg, 0.6mmol) and triethylamine (62 mg, 0.6 mmol) in EtOH (2 mL) are refluxedunder N with stirring for 2 h. The crude reaction mixture is purified ona preparative tlc plate (silica), eluting once with 3% MeOH in CHCl₃.The major band is extracted, and evaporated to dryness under reducedpressure, and the residual solid is dissolved in acetone, (5 mL),filtered, and methanesulfonic acid (32 μL, 0.5 mmol) is added slowlywith swirling. The precipitate is collected by suction filtration,rinsed with acetone and dried in a vacuum oven to give4-anilinopyrido[3,2-d]pyrimidine mesylate (91 mg, 57%) as dull yellowneedles. ¹ H NMR (DMSO) δ 11.75 (1H, slbrs), 9.11 (1H, dd, J=1.5, 4.3Hz), 8.97 (1H, s), 8.32 (1H, dd, J=1.5, 8.4 Hz), 8.12 (1H, dd, J=4.3,8.5 Hz), 7.88 (2H, d, J=8.2 Hz), 7.49 (2H, t, J=8.0 Hz), 7.32 (1H, t,J=7.0 Hz), 2.34 (3H, s).

EXAMPLE 2 4-Benzylaminopyrido[3,2-d]pyrimidine

A solution of freshly prepared 4-chloropyrido[3,2-d]pyrimidine (0.10 g,0.60 mmol) (prepared as described in the previous experimental) andbenzylamine (0.13 mL, 1.20 mmol) in propan-2-ol (15 mL) containing atrace of conc. HCl is warmed at 50° C. for 30 min, and then concentratedto dryness. The residue is partitioned between water and EtOAc, and theorganic layer worked up and chromatographed on silica gel. EtOAc elutesforeruns, while MeOH/EtOAc (1:9) elutes4-(benzylamino)pyrido[3,2-d]pyrimidine (0.11 g, 77%). ¹ H NMR (CDCl₃) δ8.67 (1H, s), 6.50 (1H, dd, J=4.3, 1.5 Hz), 8.10 (1H, dd, J=8.5, 1.5Hz), 7.63 (1H, dd, J=8.8, 4.3 Hz), 7.55 (1H, brs), 7.41-7.29 (5H, m),4.86 (2H, d, J=5.9 Hz).

EXAMPLE 3 4-(3-Bromoanilino)pyrido[3,2-d]pyrimidine

Reaction of 4-chloropyrido[3,2-d]pyrimidine (prepared as described in aprevious experimental) with 3-bromoaniline in propan-2-ol containing atrace of conc. HCl at 50° C. for 30 min, followed by chromatography ofthe product on silica gel, gives4-(3-bromophenyl)aminopyrido[3,2-d]pyrimidine (87% yield). ¹ H NMR(CDCl₃) δ 9.19 (1H, brs), 8.83 (1H, s), 8.80 (1H, dd, J=4.3, 1.5 Hz),8.29 (1H, brs), 8.19 (1H, dd, J=8.5, 1.5 Hz), 7.83 (1H, m), 7.76 (1H,dd, J=8.5, 4.3 Hz), 7.29-7.27 (2H, m).

EXAMPLE 4 4-(3-Bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine

2-cyano-6-fluoro-3-nitropyridine. A mixture of6-chloro-2-cyano-3-nitropyridine [Colbry, N. L.; Elslager, E. F. ;Werbel, L. M.; J. Het. Chem., 1984, 21, 1521-1525] (10.0 g, 0.054 mol)and KF (9.48 g, 0.163 mol) in MeCN (200 mL) is heated under reflux withstirring for 18 h, then poured into water and extracted with EtOAc. Theextract is washed with water and worked up, and the residue ischromatographed on silica gel, eluting with EtOAc/petroleum ether (3:7),to give after removal of the solvent under reduced pressure2-cyano-6-fluoro-3-nitropyridine (7.2 g, 79%). ¹ H NMR (CDCl₃) δ 8.79(1H, dd, J=9.0, 6.0 Hz), 7.48 (1H, dd, J=9.0, 3.0 Hz).

6-Fluoro-3-nitropyridine-2-carboxamide. A solution of2-cyano-6-fluoro-3-nitropyridine (1.40 g, 8.39 mmol) in 90% H₂ SO₄ (30mL) is warmed at 70° C. for 90 min, then cooled, poured onto ice andbasified with conc. ammonia. Extraction with EtOAc and workup gives6-fluoro-3-nitropyridine-2-carboxamide (0.94 g, 61%). ¹ H NMR (CDCl₃) δ8.70 (1H, dd, J=8.9, 6.5 Hz), 8.30, 8.03 (1H, 1H, brs), 7.62 (1H, dd,J=8.9, 2.9 Hz).

6-Fluoro-3H-pyrido[3,2-d]pyrimid-4-one. A solution of6-fluoro-3-nitropyridine-2-carboxamide (1.50 g, 8.10 mmol) in EtOAc (80mL) is hydrogenated over 5% Pd-C (0.30 g) at 60 psi for 2 h. Afterremoval of the catalyst by filtration, the solvent is removed underreduced pressure, to give a residue of crude3-amino-6-fluoropyridine-2-carboxamide which is used directly in thenext step. Triethyl orthoformate (60 mL) is added and the mixture isthen heated under reflux with vigorous stirring for 18 h. The cooledmixture is diluted with an equal volume of petroleum ether, and theresulting precipitate collected by filtration and is washed well withpetroleum ether to give 6-fluoro-3H-pyrido[3,2-d]pyrimid-4-one (1.26 g,84%). ¹ H NMR (DMSO) δ 12.72 (1H, brs), 8.31 (1H, dd, J=8.6, 7.7 Hz),8.20 (1H, s), 7.66 (1H, dd, J=8.6, 3.0 Hz).

4-(3-Bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine. A suspension of6-fluoro-3H-pyrido[3,2-d]pyrimid-4-one (0.20 g, 1.21 mmol) in POCl₃ (30mL) is heated under reflux with stirring until homogeneous (2 h), andthen for a further 1 h. Excess POCl₃ is removed under reduced pressure,and the residue is partitioned between CH₂ Cl₂ and saturated aqueousNaHCO₃. Workup of the organic portion gives crude4-chloro-6-fluoropyrido[3,2-d]pyrimidine (100%) as an unstable whitesolid which is used directly in the next step.

A solution of 4-chloro-6-fluoropyrido[3,2-d]pyrimidine (0.20 g, 1.1mmol) and 3-bromoaniline (0.12 mL, 2.18 mmol) in propan-2-ol (20 mL)containing conc. HCl (1 drop) is heated under reflux for 15 min, thencooled, poured into water and extracted with EtOAc. The extract isworked up, and the residue chromatographed on silica gel, eluting withEtOAc/petroleum ether (1:2)to give after removal of the solvent underreduced pressure 4-(3-bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine(0.18 g, 52%). ¹ H NMR (CDCl₃) δ 8.82 (1H, s), 8.65 (1H, brs), 8.31 (1H,t, J=7.4 Hz), 8.27 (1H, brs), 7.77 (1H, m) 7.41 (1H, dd, J=8.9, 2.2 Hz),7.29 (2H, brs).

EXAMPLE 5 4-(3-Bromoanilino)-6-chloropyrido[3,2-d]pyrimidine

6-chloro-3-nitropicolinamide. A solution of6-chloro-3-nitropicolinonitrile (1.00 g, 5.45 mmol) in 90% H₂ SO₄ (15mL) is warmed at 70° C. for 3.5 h, and then poured into ice-water. Themixture is extracted four times with EtOAc and the combined extractsworked up to give 6-chloro-3-nitropicolinamide (0.80 g, 73%). ¹ H NMR(DMSO) δ 8.55 (1H, d, J=8.5 Hz), 8.31, 8.04 (1H, 1H, 2 brs), 7.93 (1H,d, J=8.5 Hz).

6-Chloro-3H-pyrido[3,2-d]pyrimidin-4-one. A solution of6-chloro-3-nitropicolinamide (0.30 g, 1.49 mmol) in EtOAc (30 mL) ishydrogenated at 60 psi over 5% Pd-C (0.10 g) for 20 min. After removalof the catalyst by filtration the solution is concentrated to dryness togive 3-amino-6-chloropicolinamide as a yellow oil, which is useddirectly in the next step. It is dissolved in triethylorthoformate (30mL) and the mixture is heated under reflux for 18 h. Petroleum ether (30mL) is added to the cooled solution, and the resulting precipitate ofcrude 6-chloro-3H-pyrido[3,2-d]pyrimidin-4-one (0.27 g, 99%) is filteredoff and dried in a vacuum oven.

4-(3-Bromoanilino)-6-chloropyrido[3,2-d]pyrimidine. A suspension of theabove quinazolone (0.20 g, 1.10 mmol) in POCl₃ (30 mL) is heated underreflux for 3 h, and then concentrated to dryness under reduced pressure.The residue is partitioned between CH₂ Cl₂ and saturated NaHCO₃solution, and the organic portion is worked up to give4,6-dichloropyrido[3,2-d]pyrimidine (0.16 g, 73%) as a tan solid, whichis used directly in the next step. A solution of the crudedichloropyridopyrimidine (0.16 g, 0.80 mmol) and 3-bromoaniline (0.17mL, 1.60 mmol) in propan-2-ol (25 mL) containing a trace of conc. HCl iswarmed at 50° C. for 30 min. The cooled mixture is poured into saturatedNaHCO₃ and extracted with EtOAc, and the extract is worked up andchromatographed on silica gel. Elution with EtOAc/petroleum ether (1:4)gives 3-bromoaniline, while EtOAc/petroleum ether (1:1) elutes4-(3-bromoanilino)-6-chloropyrido[3,2-d]pyrimidine (0.17 g, 63%). ¹ HNMR (CDCl₃) δ 8.90 (1H, brs,) 8.84 (1H, s), 8.30 (1H, dd, J=2.1, 2.0 Hz)8.17 (1H, d, J=8.8 Hz), 7.82-7.78 (1H, m) 7.73 (1H, d, J=8.8 Hz),7.32-7.29, (2H, m).

EXAMPLE 6 4-(3-Bromoanilino)-6-aminopyrido[3,2-d]pyrimidine

Reaction of 4-(3-bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine (0.12 g,0.38 mmol)(described in a previous experimental) with a saturatedsolution of ammonia in ethanol in a pressure vessel at 100° C. for 18 hgives 6-amino-4-(3-bromoanilino)pyrido[3,2-d]pyrimidine, (87 mg, 72%). ¹H NMR (CDCl₃) δ 8.76 (1H, brs), 8.64 (1H, s), 8.23 (1H, brs), 7.93 (1H,d, J=9.0 Hz), 7.81 (1H, dt, J_(d) =7.7 Hz, J_(t) =1.8 Hz), 7.28-7.22(2H, m), 7.00 (1H, d, J=9.0 Hz), 4.90 (2H, brs).

EXAMPLE 7 4-(3-Bromoanilino)-6-methylaminopyrido[3,2-d]pyrimidine

Reaction of 4-(3-bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine (50 mg,0.16 mmol)(described in a previous experimental) with methylaminehydrochloride (32 mg, 0.47 mmol) and triethylamine (70 μL, 0.55 mmol) inethanol (10 mL) in a pressure vessel at 100° C. for 18 h gives6-methylamino-4-(3-bromoanilino)pyrido[3,2-d]pyrimidine (43 mg, 81%). ¹H NMR (CDCl₃) δ 8.81 (1H, brs), 8.61 (1H, s), 8.19 (1H, t, J=1.8 Hz),7.86 (1H, d, J=9.1 Hz,), 7.83 (1H, dt, J_(d) =7.7 Hz, J_(t) =1.8 Hz),7.28-7.21 (2H, m), 6.92 (1H, d, J=9.1 Hz), 4.97 (1H, q, J=5.0 Hz), 3.13(3H, d, J=5.0 Hz).

EXAMPLE 8 4-(3-Bromoanilino)-6-dimethylaminopyrido[3 2-d]pyrimidine

A mixture of 4-(3-bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine (0.15 g,0.47 mmol) (described in a previous experimental), dimethylaminehydrochloride (0.11 g, 1.41 mmol) and triethylamine (0.23 mL, 1.64 mmol)in EtOH (15 mL) is heated in a pressure vessel at 100° C. for 18 h. Thesolvent is removed under reduced pressure, and the residue ispartitioned between EtOAc and water. The organic portion is worked up,and the residue chromatographed on silica gel. Elution withEtOAc/petroleum ether (1:1) gives foreruns, while EtOAc elutes off4-(3-bromoanilino)-6-dimethylaminopyrido[3,2-d]pyrimidine (0.14 g, 86%).¹ H NMR (CDCl₃) δ 8.72 (1H, brs), 8.56 (1H, s), 8.17 (1H, t, J=1.9 Hz),7.85 (1H, d, J=9.3 Hz), 7.77 (1H, dt, J_(d) =7.5 Hz, J_(t) =1.9 Hz),7.27-7.18 (2H, m), 7.08 (1H, d, J=9.3 Hz), 3.21 (6H, s).

EXAMPLE 9 4-(3-Bromoanilino)-6-methoxypyrido[3,2-d]pyrimidine

4-(3-Bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine (described in aprevious experimental) (0.11 g, 0.34 mmol) is added to a solution ofNaOMe (prepared by the addition of Na metal (31 mg, 1.38 mmol) to dryMeOH (15 mL). After heating in a pressure vessel at 90° C. for 3 h, thesolution is concentrated to dryness and the residue is partitionedbetween EtOAc and water. Workup of the organic portion gives4-(3-bromophenyl)amino-6-methoxypyrido[3,2-d]pyrimidine (92 mg, 82%). ¹H NMR (CDCl₃) δ 8.73 (1H, s), 8.66 (1H, brs), 8.18 (1H, m), 8.05 (1H, d,J=8.9 Hz), 7.83-7.80 (1H, m), 7.30-7.24 (2H, m), 7.23 (1H, d, J=8.9 Hz),4.12 (3H, s).

EXAMPLE 10 4-Anilinopyrido[4,3-d]pyrimidine

4-(N-t-Butoxycarbonylamino)pyridine. To a potassium hydroxide (3.75 g,63.75 mmol), water (10 ml), and 2-methyl-2-propanol (4 mL) on ice isadded di-t-butyl-dicarbonate (6.95 g, 31.87 mmol). The resultingbiphasic solution is stirred at 25° C. for 1 week, then water (20 mL) isadded. The solution is extracted with 1× CH₂ Cl₂ and 2× EtOAc. Theorganic layer is dried (MgSO₄) and concentrated under reduced pressureto give 4-(N-t-butoxycarbonylamino)pyridine (4.08 g, 99%). ¹ H NMR(DMSO) δ 9.84 (1H, s), 8.35 (2H, d, J=6 Hz), 7.44 (2H, d, J=7 Hz), 1.49(9H, s).

4-(N-t-Butoxycarbonylamino)nicotinic acid. n-Butyl lithium (2.18M, 24mL, 52.51 mmol) is added slowly to a solution of4-(N-t-butoxycarbonylamino)-pyridine (4.08 g, 21 mmol) in THF (50 mL,stirred 0° C., stirred for 3 h, then cooled again to -78° C. and pouredinto ether (100 mL) containing dry ice. The solution is warmed to roomtemperature with constant stirring. Water is added and the mixture isneutralized with acetic acid. The resulting solid is collected by vacuumfiltration and dried in a vacuum oven to give4-(-t-butoxycarbonlamino)nicotinic acid (2.72 g, 54%) as a brown solid.¹ H NMR (DMSO) δ 11.75 (1H, brs), 8.97 (1H, s), 8.50 (1H, d, J=6.0 Hz),8.20 (1H, d, J=6.0 Hz, 1.49 (9H, s).

4-Amino nicotinic acid. A mixture of4-(N-t-butoxycarbonylamino)nicotinic acid (2.72 g, 11.4 mmol), TFA (10mL), and CH₂ Cl₂ (20 mL) is stirred at room temperature for 12 h. Thevolatiles are removed under reduced pressure, and the resulting crude4-amino nicotinic acid is used directly in the next reaction.

3H-Pyrido[4,3-d]pyrimidin-4-one. Crude 4-amino nicotinic acid (2.72 g,11.4 mmol) in formamide (20 mL) is heated to 170° C. for 12 h. Thevolatiles are distilled out under reduced pressure (0.8 mmHg). Theresidual solid is then purified on a medium pressure silica gel column,eluting with 10% MeOH in CHCl₃ to give 3H-pyrido[4,3-d]pyrimidin-4-one(780 mg, 47%) as a whitish yellow solid. 1H NMR (DMSO) δ 12.64 (1H,brs), 9.28 (1H, s), 8.83 (1H, d, J=5.5 Hz), 8.30 (1H, s), 7.58 (1H, d,J=5.8 Hz).

3H-Pyrido[4,3-d]pyrimidin-4-thione. Phosphorous pentasulfide (2.59 g,5.83 mmol) is added to a solution of 3H-pyrido[4,3-d]pyrimidin-4-one(780 mg, 5.3 mmol) in pyridine (5 mL). The mixture is refluxed for 5 h.On cooling a precipitate forms and the supernatent is decanted off. Thesolid is suspended in water (20 mL) and then filtered to yield3H-pyrido[4,3-d]pyrimidin-4-thione (676 mg, 78%) as a black solid. ¹ HNMR (DMSO) δ 14.53 (1H, brs), 9.65 (1H, s), 8.84 (1H, d, J=7.0 Hz), 8.32(1H, s), 7.64 (1H, d, J=8.0 Hz).

4-Methylthiopyrido[4,3-d]pyrimidine. A mixture of3H-pyrido[4,3-d]pyrimidin-4-thione (676 mg, 4.14 mmol), triethylamine(1.4 mL, 10.31 mmol), DMSO (4 mL), and iodomethane (0.48 mL, 7.72 mmol)is stirred for 12 h under N₂ at 25° C. The mixture is poured onto waterand extracted with EtOAc. The organic extracts are dried (MgSO₄), andthe solvent is removed under reduced pressure to yield4-methylthiopyrido[4,3-d]pyrimidine (1.15 g, quant.) as a brown solid. ¹H NMR (DMSO) δ 9.52 (1H, s), 9.16 (1H, s), 8.95 (1H, d, J=6 Hz), 7.86(1H, d, J=8 Hz), 2.75 (1H, s).

4-Anilinopyrido-[4,3-d]pyrimidine. A mixture of4-methylthiopyrido[4,3-d]pyrimidine (174 mg, 0.97 mmol), and aniline(186.2 mg, 1.99 mmol) in EtOH (2 mL) is refluxed under N₂ for 12 h.Cooling to 0° C. forms a solid which is filtered to yield4-anilinopyrido-[4,3-d]pyrimidine (34.5 mg, 16%). ¹ H NMR (DMSO) δ 10.29(1H, brs), 9.86 (1H, s), 8.82 (1H, d, J=5.8 Hz), 8.72 (1H, s), 7.85 (2H,d, J=7.5 Hz), 7.66 (1H, d, J=5.5 Hz), 7.45 (2H, t, J=8.0 Hz), 7.23 (1H,t, J=7.3 Hz).

EXAMPLE 11 4-(3-Bromoanilino)pyrido[4,3-d]pyrimidine

A mixture of 4-methylthiopyrido[4,3-d]pyrimidine (171 mg, 0.96 mmol),(see previous experimental) and 3-bromoaniline (1 mL) is heated to 100°C. for 2 h. A solid precipitates on cooling and is collected by vacuumfiltration and then recrystallized from EtOH to yield4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (30 mg, 10%). ¹ H NMR (DMSO) δ10.33 (1H. s), 9.86 (1H, s), 8.84 (1H, d, J=5.8 Hz), 8.79 (1H, s), 8.22(1H, s), 7.89 (1H, d, J=7.2 Hz), 7.69 (1H, d, J=5.8 Hz), 7.40 (2H, dt,J_(d) =8.0 Hz, J_(t) =1.5 Hz).

EXAMPLE 12 4-(3-Bromoanilino)-7-fluoropyrido[4,3-d]pyrimidine

3-Cyano-4,6-diaminopyridine. Crude 2-bromo-3-cyano-4,6-diaminopyridine[W. J. Middleton, U.S. Pat. No. 2,790,806 (Apr. 30, 1957), Du Pont;Chem. Abst. 51:P14829 (1957), see also next experimental] (15.1 g, 0.071mole) is hydrogenated in THF/MeOH (200 mL, 2:1) containing KOAc (7.0 g,0.071 mole) and 5% Pd/C (4 g) at 55 p.s.i. and 20° C. for 7 days.Filtration over celite, washing with THF/MeOH and removal of the solventgives a solid, which is dissolved in dilute HCl and water. Adjustment ofthe solution pH to 10 (conc. NaOH) and cooling gives3-cyano-4,6-diaminopyridine (6.58 g, 69%) as a yellow solid, mp 197-198°C. [Metzger, R.; Oberdorfer, J.; Schwager, C.; Thielecke, W.; Boldt, P.Liebigs Ann. Chem. 1980, 946-953 record mp (benzene) 205° C.].Extraction of the remaining liquor with EtOAc (4×200 mL) gives furtherproduct (2.12 g, 22%). ¹ H NMR (DMSO) δ 7.91 (1H, s), 6.26, 6.24 (2H,2H, brs), 5.63 (1H, s).

4,6-Diamino-3-pyridylcarboxamide. 3-Cyano-4,6-diaminopyridine (4.30 g,0.032 mole) is added to 90% H₂ SO₄ (25 mL), then stirred at 60-70° C.for 3 h. The resulting solution is added to cold conc. NaOH (40%) togive a mixture of 4,6-diamino-3-pyridylcarboxamide and inorganic salts.An analytically pure sample is obtained by chromatography on alumina(10-50% MeOH/CHCl₃) to give a pale yellow solid. ¹ H NMR (DMSO) δ 8.15(1H, s), 6.91 (2H, brs), 7.7-6.3 (2H, brm), 5.78 (2H, brs), 5.56 (1H,s).

7-Amino-4-oxo-3H-pyrido[4,3-d]pyrimidine. Crude4,6-diamino-3-pyridylcarboxamide (9.2 g) is heated in purified (EtO)₃ CH(distilled from Na, 60 mL) at 170° C. for 1.5 d. After removing thesolvent, the residue is dissolved in hot 2M NaOH, filtered, neutralized(conc. HCl) and cooled to give 7-amino-4-oxo-3H-pyrido[4,3-d]pyrimidine(3.57 g, 69% from the nitrile) as a light brown solid. ¹ H NMR (DMSO) δ11.79 (1H, brs), 8.74 (1H, s), 7.97 (1H, s), 6.76 (2H, brs), 6.38 (1H,s).

7-Fluoro-4-oxo-3H-pyrido[4,3-d]pyrimidine. A solution of7-amino-4-oxo-3H-pyrido[4,3-d]pyrimidine (1.00 g, 6.17 mmol) in 60% HBF₄(25 mL) at 0° C. is treated with solid NaNO₂ (0.85 g, 12.3 mmol, addedin portions over 2 h), and is then stirred at 0° C. for a further 1 hand at 20° C. for 30 min. The resulting mixture is ice-cooled,neutralized with saturated aqueous Na₂ CO₃, and extracted with EtOAc(4×100 mL). The extract is washed with water, then filtered throughsilica gel (EtOAc) to give 7-fluoro-4-oxo-3H-pyrido[4,3-d]pyrimidine(0.48 g, 47%) as a cream solid. ¹ H NMR (DMSO) δ 12.69 (1H, brs), 9.01(1H, s), 8.31 (1H, s), 7.34 (1H, s)

4-(3-Bromoanilino)-7-fluoropyrido[4,3-d]pyrimidine. A suspension of7-fluoro-4-oxo-3H-pyrido[4,3-d]pyrimidine (0.23 g, 1.39 mmol) in POCl₃(10 mL) is stirred under reflux for 3.5 h, and is then concentratedunder vacuum. The resulting oil is ice-cooled, diluted with CH₂ Cl₂ (100mL), saturated aqueous Na₂ CO₃ (40 mL) and ice, and stirred at 20° C.for 2 h. The CH₂ Cl₂ extract is separated and the aqueous portionfurther extracted with CH₂ Cl₂ (2×100 mL), and then the combinedextracts are dried (Na₂ SO₄) and filtered to give crude4-chloro-7-fluoropyrido[4,3-d]pyrimidine. 3-Bromoaniline (1.26 g, 7.35mmole), 3-bromoaniline hydrochloride (20 mg) and dry isopropanol (5 mL)are added, then the resulting solution is concentrated under vacuum toremove the CH₂ Cl₂ and stirred at 20° C. for 1 h. Upon addition ofdilute NaHCO₃ and water, the product crystallises. Filtration, washingwith water and CH₂ Cl₂, gives pure4-(3-bromoanilino)-7-fluoropyrido[4,3-d]pyrimidine (297 mg, 67%) as acream solid. ¹ H NMR (DMSO) δ 10.38 (1H, brs), 9.59 (1H, s), 8.72 (1H,s), 8.17 (1H, s), 7.85 (1H, m), 7.38 (3H, m)

EXAMPLE 13 7-Amino-4-anilinopyrido[4,3-d]pyrimidine

4,6-Diamino-2-bromo-3-cyanopyridine. HBr is bubbled for 2 h into amixture of malononitrile (16.3 g, 0.247 mol) and toluene (400 mL) at 0°C. A light yellow precipitate forms. The reaction mixture is then heatedat 100° C. for 2 h, with much gas evolution. After cooling to roomtemperature, the yellow solid is isolated via suction filtration, washedwith toluene and air dried. The solid (25.96 g) is mixed with water (500mL), and the pH of the suspension is adjusted to 9˜10 with NH₄ OH (conc.˜15 mL). After stirring at room temperature for 1 h, the mixture isfiltered. Recrystallization from EtOH affords a yellow solid. Afterdrying at 60° C. in a vacuum oven, 4,6-diamino-2-bromo-3-cyanopyridine(12.95 g, 49%) is obtained. ¹ H NMR (DMSO) δ 6.67 (2H, brs), 6.55 (2H,brs), 5.59 (1H, s).

2,4-Diamino-5-cyanopyridinium acetate.4,6-Diamino-2-bromo-3-cyanopyridine (12.77 g, 60 mmol) is hydrogenatedin THF/MeOH (240 mL, 2:1) containing KOAc (5.9 g, 60 mmol) and 20% Pd/C(0.5 g) at 18 psi at 25° C. for 4 h. The mixture is celite filtered andthe solvent is stripped under reduced pressure to give a solid (11.15 g)which is stirred with THF (100 mL) at room temperature for 20 min. Themixture is refiltered and the filtrate is stripped to dryness to givethe desired product. After drying in a vacuum oven,2,4-diamino-5-cyanopyridinium acetate (10.65 g, 92%) is collected as ayellow solid. ¹ H NMR (DMSO) δ 7.90 (1H, s), 6.26 (4H, brs), 5.62 (1H,s), 1.90 (3H, s).

7-Amino-4-thiono-3H-pyrido[4,3-d]pyrimidine. A mixture of2,4-diamino-5-cyanopyridinium acetate (0.199 g, 1.0 mmol), triethylorthoformate (1.95 mL) and Ac₂ O (1.95 mL) is refluxed under N₂ withstirring for 3 h. The solvent is then stripped and the residue isdissolved in MeOH (10 mL) containing NaOMe (0.81 g, 15 mmol). H₂ S isbubbled through the mixture for ˜5 min. which is then refluxedovernight. After the solvent is stripped, the residue is dissolved inhot water and boiled with charcoal. After filtration, the filtrate isneutralized with acetic acid whilst hot to generate a yellow solid. Oncooling, the solid is collected by suction filtration, and is dried in avacuum oven overnight. 7-Amino-4-thiono-3H-pyrido[4,3-d]pyrimidine (84mg, 51%) is isolated as light yellow solid. ¹ H NMR (DMSO) δ 9.82 (1H,s), 9.34 (1H, s), 8.37 (1H, s), 7.80 (2H, d, J=7.5 Hz), 7.38 (2H, t,J=7.5 Hz), 7.12 (1H, t, J=7.5 Hz), 6.61 (2H, brs) 6.43 (1H, s).

7-Amino-4-methylthiopyrido[4,3-d]pyrimidine. NEt₃ (6 mL, 43 mmol) isadded to a solution of 7-amino-4-thiono-3H-pyrido[4,3-d]pyrimidine (0.77g, 4.3 mmol) in DMSO (7 mL) stirred under N₂ at 25° C. After the twophases have been stirred for 20 min, MeI (0.26 mL, 4.2 mmol) is added.After 2 h, the reaction mixture is poured onto stirring ice-water. Solidforms instantly. After further cooling at 0° C., the solid is collectedby suction filtration and dried in a vacuum oven to give7-amino-4-methylthiopyrido[4,3-d]pyrimidine (0.564 g, 68%). ¹ H NMR(DMSO) δ 8.98 (1H, s), 8.71 (1H, s), 6.94 (2H, brs), 6.49 (1H, s) 2.63(3H, s).

7-Amino-4-anilinopyrido[4,3-d]pyrimidine. A mixture of7-amino-4-methylthiopyrido[4,3-d]pyrimidine (0.136 g, 0.7 mmol) andaniline (0.5 mL, 5.5 mmol) is refluxed under N₂ at 180° C. for 2 h. Thereaction mixture is cooled to 25° C., when it precipitates. The solid iscollected by suction filtration and recrystallized from isopropanol, anddried in a vacuum oven overnight.7-Amino-4-anilinopyrido[4,3-d]pyrimidine (84 mg, 51%) is isolated as alight yellow solid. ¹ H NMR (DMSO) δ 9.82 (1H, s), 9.34 (1H, s), 8.37(1H, s), 7.80 (2H, d, J=7.5 Hz), 7.38 (2H, t, J=7.5 Hz), 7.12 (1H, t,J=7.5 Hz), 6.61 (2H, brs) 6.43 (1H, s).

EXAMPLE 14 7-Amino-4-(3-hydroxyanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (299 mg, 1.56mmole) and 3-aminophenol (1.60 g, 14.7 mmole) is stirred at 160° C. for15 min. The resulting product is chromatographed over silica gel (9%MeOH/CH₂ Cl₂) to give7-amino-4-(3-hydroxyanilino)pyrido[4,3-d]pyrimidine (108 mg, 18%) as apale orange solid. ¹ H NMR (DMSO) δ 9.69 (1H, brs), 9.44 (1H, brs), 9.33(1H, s), 8.38 (1H, s), 7.37 (1H, t, J=2.1 Hz), 7.21 (1H, brd, J=8.4 Hz),7.14 (1H, t, J=8.0 Hz), 6.59 (2H, brs), 6.53 (1H, ddd, J=7.9, 2.2, 0.8Hz), 6.43 (1H, s).

EXAMPLE 15 7-Amino-4-(3-methoxyanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (226 mg, 1.18mmol) (described in the previous experimental) and m-anisidine (1.00 mL,8.90 mmol) is stirred under N₂ at 190° C. for 1.5 h. The resultingproduct is chromatographed over silica gel (5-7% EtOH/EtOAc) to give7-amino-4-(3-methoxyanilino)pyrido[4,3-d]pyrimidine (136 mg, 43%) as alight brown solid. ¹ H NMR (DMSO) δ 9.78 (1H, brs), 9.34 (1H, s), 8.40(1H, s), 7.50 (1H, brs), 7.44 (1H, d, J=8.0 Hz), 7.28 (1H, t, J=8.2 Hz),6.71 (1H, dd, J=8.2, 2.3 Hz), 6.61 (2H, brs), 6.45 (1H, s), 3.77 (3H,s).

EXAMPLE 16 7-Amino-4-(2-methoxyanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (227 mg, 1.18mmole) and o-anisidine (1.00 mL, 8.87 mmol) is stirred under N₂ at 180°C. for 2.5 h. The resulting product is chromatographed over silica gel(5% EtOH/EtOAc) to give7-amino-4-(2-methoxyanilino)pyrido[4,3-d]pyrimidine (147 mg, 47%) as ayellow solid. ¹ H NMR (DMSO) δ 9.44 (1H, brs), 9.25 (1H, s), 8.22 (1H,s), 7.54 (1H, dd, J=7.7, 1.4 Hz), 7.24 (1H, ddd, J=8.1, 7.4, 1.5 Hz),7.10 (1H, dd, J=8.2, 1.2 Hz), 6.98 (1H, dt, J_(d) =1.3 Hz, J_(t) =7.5Hz), 6.52 (2H, brs), 6.41 (1H, s), 3.79 (3H, s).

EXAMPLE 17 7-Amino-4-(3-aminoanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (307 mg, 1.60mmol) (described in a previous experimental) and 3-nitroaniline (2.00 g,14.5 mmol) is stirred at 200° C. for 1.5 h, and the crude product issuspended in MeOH/THF (4:1, 250 mL) and hydrogenated over 5% Pd/C (2 g)at 60 psi and 20° C. for 24 h. The solution is filtered over celite,washing thoroughly (hot MeOH), and is then absorbed onto alumina andchromatographed on alumina (4-8% EtOH/CHCl₃) to give7-amino-4-(3-aminoanilino)pyrido[4,3-d]pyrimidine (66 mg, 16%) as agreen solid. ¹ H NMR (DMSO) δ 9.57 (1H, brs), 9.30 (1H, s), 8.33 (1H,s), 7.04 (1H, t, J=2.0 Hz), 6.99 (1H, t, J=8.0 Hz), 6.88 (1H, brd, J=8.0Hz), 6.55 (2H, brs), 6.40 (1H, s), 6.34 (1H, dd, J=7.9, 1.3 Hz), 5.10(2H, brs).

EXAMPLE 18 7-Amino-4-(4-aminoanilino)pyrido[4,3-d]pyrimidine

7-Amino-4-(4-acetamidoanilino)pyrido[4,3-d]pyrimidine. A mixture of7-amino-4-methylthiopyrido[4,3-d]pyrimidine (138 mg, 0.72 mmole) and4-aminoacetanilide (1.50 g, 10.0 mmole) is stirred under N₂ at 200° C.for 1 h. The resulting product is chromatographed over alumina (8-10%MeOH/CH₂ Cl₂) to give7-amino-4-(4-acetamidoanilino)pyrido[4,3-d]pyrimidine (110 mg, 52%) as apale yellow solid. ¹ H NMR (DMSO) δ 9.94, 9.79 (1H, 1H, 2 brs), 9.31(1H, s), 8.34 (1H, s), 7.69 (2H, d, J=8.9 Hz), 7.57 (2H, d, J=8.9 Hz),6.57 (2H, brs), 6.43 (1H, s), 2.05 (3H, s).

7-Amino-4-(4-aminoanilino)pyrido[4,3-d]pyrimidine. A solution of7-amino-4-(4-acetamidoanilino)pyrido[4,3-d]pyrimidine (0.30 g, 1.02mmole) in aqueous NaOH (2M, 10 mL) and MeOH (10 mL) is stirred at 100°C. for 7 h. The resulting product is chromatographed over alumina (3-4%EtOH/CHCl₃) to give 7-amino-4-(4-aminoanilino)pyrido[4,3-d]pyrimidine(86 mg, 33%) as an orange solid. ¹ H NMR (DMSO) δ 9.58 (1H, brs), 9.24(1H, s), 8.25 (1H, s), 7.31 (2H, d, J=8.6 Hz), 6.58 (2H, d, J=8.6 Hz),6.48 (2H, brs), 6.39 (1H, s), 5.00 (2H, brs).

EXAMPLE 19 7-Amino-4-(3-dimethylaminoanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (245 mg, 1.28mmol) (described in a previous experimental) andN,N-dimethyl-1,3-phenylenediamine (1.60 g, 11.8 mmol) is stirred underN₂ at 190° C. for 1 h, and the resulting product is chromatographed(twice) over alumina (3% EtOH/CHCl₃) to give7-amino-4-(3-dimethylaminoanilino)pyrido[4,3-d]pyrimidine (113 mg, 32%)as a pale yellow solid. ¹ H NMR (DMSO) δ 9.66 (1H, brs), 9.33 (1H, s),8.36 (1H, s), 7.22 (1H, brd, J=7.8 Hz), 7.16 (2H, m), 6.57 (2H, brs),6.51 (1H, ddd, J=8.0, 2.3, 1.2 Hz), 6.42 (1H, s), 2.91 (6H, s).

EXAMPLE 20 7-Amino-4-(4-dimethylaminoanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (256 mg, 1.33mmole) and N,N-dimethyl-1,4-phenylenediamine (1.95 g, 14.4 mmole) isstirred under N₂ at 190° C. for 20 min. The resulting product ischromatographed over alumina (3-7% EtOH/CHCl₃) to give7-amino-4-(4-dimethylaminoanilino)pyrido[4,3-d]pyrimidine (198 mg, 53%)as an orange solid. ¹ H NMR (DMSO) δ 9.67 (1H, brs), 9.27 (1H, s), 8.27(1H, s), 7.51 (2H, d, J=8.9 Hz), 6.75 (2H, d, J=8.9 Hz), 6.51 (2H, brs),6.39 (1H, s), 2.89 (6H, s).

EXAMPLE 21 7-Amino-4-(2-nitroanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (220 mg, 1.15mmole) and 2-nitroaniline (2.00 g, 14.5 mmole) is heated to 100° C.,then excess dry HCl gas is added to the hot stirred solution, and themixture stirred at 160° C. for 20 min. The resulting product isneutralized with excess NaHCO₃, dissolved in MeOH/CHCl₃, dried ontosilica gel and chromatographed over silica gel (2-4% MeOH/CH₂ Cl₂) togive 7-amino-4-(2-nitroanilino)pyrido[4,3-d]pyrimidine (108 mg, 33%) asa yellow brown solid. ¹ H NMR (DMSO) δ 10.40 (1H, brs), 9.24 (1H, brs),8.20 (1H, brs), 8.12 (1H, brs), 8.01 (2H, brs), 7.75 (1H, brs), 6.70(2H, brs), 6.43 (1H, brs).

EXAMPLE 22 7-Amino-4-(3-nitroanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (127 mg, 0.66mmol) (described in a previous experimental) and 3-nitroaniline (1.70 g,12.3 mmol) is stirred under N₂ at 200° C. for 1.5 h. The resultingproduct is chromatographed over alumina (5-20% EtOH/CHCl₃) to give7-amino-4-(3-nitroanilino)pyrido[4,3-d]pyrimidine (81 mg, 39%) as abrown solid. ¹ H NMR (DMSO) δ 10.17 (1H, brs), 9.37 (1H, s), 8.87 (1H,brs), 8.48 (1H, s), 8.33 (1H, brd, J=7.5 Hz), 7.95 (1H, ddd, J=8.2, 2.1,1.0 Hz), 7.67 (1H, t, J=8.2 Hz), 6.70 (2H, brs), 6.47 (1H, s).

EXAMPLE 23 7-Amino-4-(3-fluoroanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (215 mg, 1.12mmol) and 3-fluoroaniline (1.16 g, 10.4 mmol) is stirred at 160° C. for30 min. The resulting product is chromatographed over silica gel (6-7%MeOH/CH₂ Cl₂) to give 7-amino-4-(3-fluoroanilino)pyrido[4,3-d]pyrimidine(185 mg, 65%) as a white solid. ¹ H NMR (DMSO) δ 9.94 (1H, brs), 9.36(1H, s), 8.46 (1H, s), 7.91 (1H, brd, J=11.9 Hz), 7.63 (1H, brd, J=8.1Hz), 7.41 (1H, dd, J=15.7, 7.7 Hz), 6.93 (1H, dt, J_(t) =8.5 Hz, J_(d)=2.4 Hz), 6.68 (2H, brs), 6.38 (1H, s).

EXAMPLE 24 7-Amino-4-(3-chloroanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (208 mg, 1.08mmol) and 3-chloroaniline (1.21 g, 9.48 mmol) is stirred at 150° C. for20 min. The resulting product is chromatographed over alumina (5-10%MeOH/CH₂ Cl₂) to give 7-amino-4-(3-chloroanilino)pyrido[4,3-d]pyrimidine(177 mg, 60%) as a white solid. ¹ H NMR (DMSO) δ 9.92 (1H, brs), 9.35(1H, s), 8.45 (1H, s), 8.08 (1H, brs), 7.79 (1H, brd, J=8.0 Hz), 7.40(1H, t, J=8.1 Hz), 7.16 (1H, dd, J=7.9, 1.3 Hz), 6.68 (2H, brs), 6.46(1H, s).

EXAMPLE 25 7-Amino-4-(3,4-dichloroanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (247 mg, 1.29mmol) and 3,4-dichloroaniline (1.50 g, 9.26 mmol) is stirred at 165° C.for 30 min. The resulting product is chromatographed over silica gel(7-8% MeOH/CH₂ Cl₂) to give7-amino-4-(3,4-dichloroanilino)pyrido[4,3-d]pyrimidine (252 mg, 64%) asa pale yellow solid. ¹ H NMR (DMSO) δ 9.97 (1H, brs), 9.34 (1H, s), 8.47(1H, s), 8.29 (1H, brs), 7.86 (1H, brd, J=8.6 Hz), 7.62 (1H, d, J=8.8Hz), 6.70 (2H, brs), 6.46 (1H, s).

EXAMPLE 26 7-Amino-4-(2-bromoanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (198 mg, 1.03mmol) (described in a previous experimental) and 2-bromoaniline (1.00mL, 9.18 mmol) is stirred under N₂ at 180° C. for 2.5 h, and theresulting product is chromatographed on alumina (1% EtOH/CHCl₃) to give7-amino-4-(2-bromoanilino)pyrido[4,3-d]pyrimidine (108 mg, 33%) as apale yellow solid, ¹ H NMR (DMSO) δ 9.91 (1H, brs), 9.27 (1H, s), 8.20(1H, s), 7.73 (1H, d, J=7.9 Hz), 7.50 (1H, m), 7.44 (1H, t, J=6.9 Hz),7.25 (1H, m), 6.59 (2H, brs), 6.42 (1H, s).

EXAMPLE 27 7-Amino-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (167 mg, 0.87mmol) (described in a previous experimental) and 3-bromoaniline (0.75mL, 7.8 mmol) is stirred under N₂ at 190° C. for 2.5 h, and theprecipitate which appears on cooling is recrystallized from Pr^(i) OH. ¹H NMR (DMSO) δ 9.91 (1H, brs), 9.34 (1H, s), 8.45 (1H, s), 8.19 (1H, s),7.84 (1H, d, J=8.0 Hz), 7.34 (1H, t, J=8.0 Hz), 7.29 (1H, d, J=8.2 Hz),6.68 (2H, brs), 6.45 (1H, s).

EXAMPLE 28 7-Amino-4-(4-bromoanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (261 mg, 1.36mmole) and 4-bromoaniline (1.00 g, 5.81 mmole) is stirred under N₂ at200° C. for 15 min. The resulting product is chromatographed on silicagel (10-15% EtOH/EtOAc) to give7-amino-4-(4-bromoanilino)pyrido[4,3-d]pyrimidine (200 mg, 46%) as apale yellow solid. ¹ H NMR (DMSO) δ 9.88 (1H, brs), 9.34 (1H, s), 8.40(1H, s), 7.83 (2H, d, J=8.8 Hz, 7.55 (2H, d, J=8.8 Hz), 6.64 (2H, brs),6.44 (1H, s).

EXAMPLE 29 7-Amino-4-(3-iodoanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (72 mg, 0.37mmol) and 3-iodoaniline (1.25 g, 5.71 mmol) is stirred at 160° C. for 30min. The resulting product is chromatographed over silica gel (5-7%MeOH/CH₂ Cl₂) to give 7-amino-4-(3-iodoanilino)pyrido[4,3-d]pyrimidine(83 mg, 61%) as a light brown rosettes. ¹ H NMR (DMSO) δ 9.84 (1H, brs),9.34 (1H, s), 8.44 (1H, s), 8.30 (1H, brs), 7.90 (1H, dd, J=7.9, 0.8Hz), 7.47 (1H, d, J=7.7 Hz), 7.18 (1H, t, J=8.0 Hz), 6.66 (2H, brs),6.46 (1H, s).

EXAMPLE 30 7-Amino-4-(2-trifluoromethylanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (300 mg, 1.56mmol), 2-aminobenzotrifluoride hydrochloride (1.00 g, 5.06 mmol) and2-aminobenzotrifluoride (2.00 g, 12.4 mmol) is stirred at 160° C. for 10min. The resulting product is neutralized with excess NaHCO₃, dissolvedin MeOH/CHCl₃, dried onto silica gel and chromatographed over silica gel(6-7% MeOH/CH₂ Cl₂) to give7-amino-4-(2-trifluoromethylanilino)pyrido[4,3-d]pyrimidine (194 mg,41%) as a cream solid, mp (MeOH/CHCl₃ /light petroleum) 126-130° C.(dec.). ¹ H NMR (DMSO) δ 10.60 (1H, brs), 9.17 (1H, brs), 8.13 (1H,brs), 7.76, 7.69 (1H, 1H, m, m), 7.45 (2H, m), 6.66 (2H, brs), 6.36 (1H,s).

EXAMPLE 31 7-Amino-4-(3-trifluoromethylanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (234 mg, 1.22mmol) (described in a previous experimental) and 3-aminobenzotrifluoride(2.00 mL, 16.0 mmol) is stirred under N₂ at 190-200° C. for 2 h, and theresulting product is then chromatographed over silica gel (5-10%EtOH/EtOAc), and then over alumina (5-7% EtOH/CHCl₃) to give7-amino-4-(3-trifluoromethylanilino)pyrido[4,3-d]pyrimidine (157 mg,42%) as a cream solid. ¹ H NMR (DMSO) δ 10.04 (1H, s), 9.37 (1H, s),8.46 (1H, s), 8.31 (1H, s), 8.19 (1H, d, J=8.2 Hz), 7.62 (1H, t, J=8.0Hz), 7.45 (1H, d, J=7.7 Hz), 6.69 (2H, brs), 6.47 (1H, s).

EXAMPLE 32 7-Amino-4-(4-trifluoromethylanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (390 mg, 2.03mmol), 4-aminobenzotrifluoride hydrochloride (0.40 g, 2.02 mmol) and4-aminobenzotrifluoride (1.61 g, 10.0 mmol) is stirred at 180° C. for 2min. The resulting product is neutralized with excess NaHCO₃, dissolvedin MeOH/CHCl₃, dried onto alumina and chromatographed over alumina (4-7%MeOH/CH₂ Cl₂) to give7-amino-4-(4-trifluoromethylanilino)pyrido[4,3-d]pyrimidine (390 mg,63%) as a cream solid. Analytically pure material was obtained byfurther chromatography over silica gel (5% MeOH/CH₂ Cl₂) to give paleyellow needles. ¹ H NMR (DMSO) δ 10.09 (1H, brs), 9.40 (1H, s), 8.48(1H, s), 8.13 (2H, d, J=8.2 Hz), 7.74 (2H, d, J=8.7 Hz), 6.72 (2H, brs),6.40 (1H, s)

EXAMPLE 33 4-(3-Bromoanilino)-7-methylaminopyrido[4,3-d]pyrimidine

A mixture of 7-fluoro-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (74 mg,0.23 mmol), triethylamine (7 mL, 50 mmol) and methylamine hydrochloride(3.0 g, 44 mmol) in isopropanol (30 mL) contained in a steel bomb isstirred at 95° C. (oil bath) for 5 h. The resulting mixture isconcentrated under vacuum, basified with aqueous Na₂ CO₃, diluted withwater and extracted with EtOAc (3×100 mL). Chromatography of thisextract on silica gel (3% MeOH/CH₂ Cl₂) gives4-(3-bromoanilino)-7-methylaminopyrido[4,3-d]pyrimidine (50 mg, 65%) asa pale yellow solid. ¹ H NMR (DMSO) δ 9.93 (1H, brs), 9.37 (1H, s), 8.47(1H, s), 8.18 (1H, s), 7.84 (1H, d, J=7.8 Hz), 7.34 (1H, t, J=7.9 Hz),7.30 (1H, brd, J=8.1 Hz), 7.19 (1H, q, J=4.7 Hz), 6.35 (1H, s), 2.85(3H, d, J=4.8 Hz).

EXAMPLE 34 4-(3-Bromoanilino)-7-dimethylaminopyrido[4,3-d]pyrimidine

A mixture of 7-fluoro-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (101 mg,0.32 mmol), triethylamine (4.4 mL, 32 mmole) and dimethylaminehydrochloride (2.58 g, 32 mmol) in isopropanol (30 mL) contained in asteel bomb is stirred at 100° C. (oil bath) for 4 h. The resultingsolution is concentrated under vacuum, basified with aqueous Na₂ CO₃ anddiluted with water to give a solid. Filtration and recrystallisationfrom MeOH/CHCl₃ gives7-dimethylamino-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (102 mg, 94%)as a pale yellow solid. ¹ H NMR (DMSO) δ 9.93 (1H, brs), 9.42 (1H, s),8.48 (1H, s), 8.19 (1H, s), 7.85 (1H, d, J=7.7 Hz), 7.35 (1H, t, J=7.9Hz), 7.30 (1H, brd, J=7.8 Hz), 6.53 (1H, s), 3.16 (6H, s).

EXAMPLE 354-[N-(3-Bromophenyl)-N-methylamino]-7-methylaminopyrido[4,3-d]pyrimidine

A mixture of 7-fluoro-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (100 mg,0.31 mmole), triethylamine (4.4 mL, 32 mmole) and methylaminehydrochloride (2.12 g, 32 mmole) in isopropanol (30 mL) contained in asteel bomb is stirred at 100° C. (oil bath) for 5 h. The resultingmixture is concentrated under vacuum, basified with aqueous Na₂ CO₃,diluted with water and extracted with EtOAc (3×100 mL). Chromatographyof this extract on silica gel (1-2% MeOH/CH₂ Cl₂) gives4-[N-(3-bromophenyl)-N-methylamino]-7-methylaminopyrido[4,3-d]pyrimidine(23 mg, 21%) as a pale yellow solid. ¹ H NMR (DMSO) δ 8.14 (1H, s), 7.79(1H, s), 7.30 (1H, t, J=8.0 Hz), 7.20 (1H, ddd, J=7.9, 1.8, 0.8 Hz),7.03 (1H, brq, J=4.9 Hz), 7.01 (1H, t, J=1.9 Hz), 6.82 (1H, ddd, J=7.8,1.8, 0.9 Hz), 6.25 (1H, s), 3.40 (3H, s), 2.73 (3H, d, J=4.9 Hz).

EXAMPLE 36 7-Acetylamino-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (0.154 g,0.49 mmol), acetic anhydride (0.14 mL, 1.5 mmol), triethylamine (0.14mL, 1.0 mmol) and a catalytic amount of 4-(N,N-dimethylamino)pyridineare stirred under N₂ at room temperature for 18 h. The reaction is thenquenched by addition of ice water. The dark precipitate is collected byBuchner filtration and is purified by preparative tlc (Rf=0.25, 7%MeOH/CHCl₃). Recrystallization from EtOH gives7-acetylamino-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (13.5 mg, 7.7%).¹ H NMR (DMSO) δ 10.92 (1H, s), 10.22 (1H, s), 9.64 (1H, s), 8.70 (1H,s), 8.28 (1H, s), 8.21 (1H, s), 7.88 (1H, d, J=7.7 Hz) 7.41-7.34 (3H,m), 2.16 (3H, s).

EXAMPLE 37 4-(3-Bromoanilino)-7-methoxypyrido[4,3-d]pyrimidine

A solution of 7-fluoro-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (100mg, 0.31 mmol) in 1M sodium methoxide-methanol (30 mL) is stirred underreflux for 42 h. The resulting mixture is concentrated under reducedpressure, diluted with water and neutralized with dilute HCl to give7-methoxy-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (92 mg, 89%) as awhite solid. ¹ H NMR (DMSO) δ 10.22 (1H, brs), 9.57 (1H, s), 8.63 (1H,s), 8.19 (1H, s), 7.86 (1H, brd, J=7.9 Hz), 7.39 (1H, t, J=7.9 Hz), 7.35(1H, dd, J=7.9, 1.5 Hz), 6.96 (1H, s), 4.00 (3H, s).

EXAMPLE 38 4-Benzylaminopyrido[4,3-d]pyrimidine

4-Methylthiopyrido[4,3-d]pyrimidine (160.4 mg, 0.902 mmol), andbenzylamine (106.3 mg, 0.992 mmol) in EtOH (2 mL) are heated at 80° C.for 12 h, and then the solvent is removed under reduced pressure. Theresulting solid is suspended in CH₂ Cl₂, filtered, and the resultingsolid is purified by preparative tlc on silica, eluting with 5% MeOH inCHCl₃. Removal of the solvent under reduced pressure yields4-benzylaminopyrido[4,3-d]pyrimidine (36 mg, 17%). ¹ H NMR (DMSO) δ 9.60(1H, s), 9.37 (1H, t, J=5.8 Hz), 8.72 (1H, d, J=5.8 Hz), 8.57 (1H, s),7.54 (1H, d, J=5.8 Hz), 7.37 (2H, d, J=7.0 Hz), 7.33 (2H, t, J=7.3 Hz),7.25 (1H, t, J=7.2 Hz), 4.81 (2H, d, J=5.8 Hz).

EXAMPLE 39 4-([R]-1-Phenylethylamino)pyrido[4,3-d]pyrimidine

To a mixture of 4-methylthiopyrido[4,3-d]pyrimidine (85 mg, 0.48 mmol)and EtOH (2.5 mL) is added R-methylbenzylamine (0.13 mL, 1.0 mmol)dropwise. The resulting mixture is refluxed at 80° C. for 20 h. Thesolvent is removed under reduced pressure to give an oil which iscrystallized from MeOH to give4-([R]-1-phenylethylamino)pyrido[4,3-d]pyrimidine (41.6 mg, 35%), mp138-138.5° C. ¹ H NMR (DMSO) δ 9.77 (1H, d, J=0.7 Hz), 9.00 (1H, d,J=7.7 Hz), 8.73 (1H, d, J=5.8 Hz), 8.54 (1H, s), 7.53 (1H, dd, J=5.8,0.5 Hz), 7.45 (2H, d, J=7.2 Hz), 7.33 (2H, t, J=7.6 Hz), 7.23 (1H, tt,J=7.5, 1.2 Hz), 5.63 (1H, p, J=7.2 Hz), 1.61 (3H, t, J=7.0 Hz).

EXAMPLE 40 7-Amino-4-benzylaminopyrido[4,3-d]pyrimidine

A mixture of 2,4-diamino, 5-cyanopyridinium acetate (8.78 g, 45 mmol),formic acid (10.66 g, 0.204 mol) and benzylamine (45 mL, 0.41 mol) isheated at 200° C. under N₂ for 2 h. Upon cooling, it solidifies. Water(500 mL) is added and the gummy solid/water mixture is stirred for ˜20min. at 0 C. The liquid is decanted. The solid is washed with water andthen recrystallized from isopropanol (25 mL). After drying in a vacuumoven overnight, 7-amino-2-benzylaminopyrido[4,3-d]pyrimidine (8.29 g,73%) is obtained as a light yellow solid. ¹ H NMR (DMSO) δ 9.10 (1H, s),8.85 (1H, t, J=5.8 Hz), 8.25 (1H, s), 7.21-7.36 (5H, m), 6.46 (2H, brs),6.35 (1H, s), 4.74 (2H, d, J=6.0 Hz).

EXAMPLE 41 7-Amino-4-([R]-1-phenylethylamino)pyrido[4,3-d]pyrimidine

A mixture of [R]-1-phenylethylamine (0.072 mL, 0.55 mmol) and7-amino-4-methylthiopyrido[4,3-d]pyrimidine (97 mg, 0.5 mmol) (describedin a previous experimental) is heated at 180° C. under N₂ for 1.5 hr.The reaction is then cooled to room temperature producing a precipitate.The mixture is added to water and CHCl₃, sonicated and filtered. Thephases are separated and the aqueous phase is extracted with CHCl₃. Thecombined extracts are washed with water, saturated brine and dried(MgSO₄). The solvent is removed under reduced pressure and the residuepurified by using preparative tlc (5% MeOH/CHCl₃) and recrystallizationfrom CHCl₃ to give7-amino-4-([R]-1-phenylethylamino)pyrido[4,3-d]pyrimidine (14.5 mg,11%), mp 231.8-232.1° C. ¹ H NMR (DMSO) δ 9.23 (1H, s), 8.50 (1H, d,J=8.0 Hz), 8.19 (1H, s), 7.41 (2H, d, J=7.0 Hz), 7.31 (2H, t, J=8.0 Hz,7.21 (1H, tt, J=7.4, 1.2 Hz), 6.45 (2H, s), 6.33 (1H, s), 5.56 (1H, p,J=7.2 Hz), 1.55 (3H, d, J=7.0 Hz).

EXAMPLE 42 7-Amino-4-(2-aminobenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (136 mg, 0.71mmol) (described in a previous experimental) and 2-aminobenzylamine(1.70 g, 13.8 mmol) in isopropanol (5 mL) is stirred at reflux for 1 h,and the resulting product is chromatographed on silica gel (7-20%EtOH/EtOAc) and alumina (6-10% EtOH/CHCl₃) to give7-amino-4-(2-aminobenzylamino)pyrido[4,3-d]pyrimidine (89 mg, 47%) as awhite solid. ¹ H NMR (DMSO) δ 9.08 (1H, s), 8.68 (1H, t, J=5.8 Hz), 8.26(1H, s), 7.05 (1H, d, J=7.4 Hz), 6.96 (1H, t, J=7.6 Hz), 6.63 (1H, d,J=7.9 Hz), 6.51 (1H, t, J=7.4 Hz), 6.46 (2H, brs), 6.35 (1H, s), 5.20(2H, brs), 4.56 (2H, d, J=5.8 Hz).

EXAMPLE 43 7-Amino-4-(3-dimethylaminobenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (236 mg, 1.23mmol) (described in a previous experimental) and3-dimethylamino-benzylamine (1.36 g, 9.07 mmol) in isopropanol (5 mL) isstirred under N₂ at reflux for 1 h, and the resulting product ischromatographed on silica gel (10-15% EtOH/EtOAc), then on alumina (1%EtOH/CHCl₃) to give7-amino-4-(3-dimethylaminobenzylamino)pyrido[4,3-d]pyrimidine (145 mg,40%) as a white solid. ¹ H NMR (DMSO) δ 9.11 (1H, s), 8.79 (1H, t, J=5.9Hz), 8.26 (1H, s), 7.11 (1H, dd, J=8.0, 7.7 Hz), 6.73 (1H, brs), 6.63(1H, d, J=7.6 Hz), 6.60 (1H, dd, J=8.1, 2.2 Hz), 6.44 (2H, brs), 6.35(1H, s), 4.67 (2H, d, J=5.8 Hz), 2.86 (6H, s).

EXAMPLE 44 7-Amino-4-(3-nitrobenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (228 mg, 1.19mmol) (described in a previous experimental) and 3-nitrobenzylamine(0.81 g, 5.33 mmol) is stirred under N₂ at 150-160° C. for 1.5 h, andthe resulting product chromatographed on silica gel (5-10% EtOH/EtOAc)to give 7-amino-4-(3-nitrobenzylmino)pyrido[4,3-d]pyrimidine (151 mg,43%) as a yellow solid. ¹ H NMR (DMSO) δ 9.11 (1H, s), 8.98 (1H, t,J=5.5 Hz), 8.26 (1H, s), 8.22 (1H, brs), 8.12 (1H, dd, J=8.0, 1.8 Hz),7.83 (1H, d, J=7.7 Hz), 7.63 (1H, t, J=7.9 Hz), 6.50 (2H, brs), 6.38(1H, s), 4.85 (2H, d, J=5.8 Hz).

EXAMPLE 45 7-Amino-4-(3-methoxybenzylamino)-pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (136 mg, 0.71mmol) (described in a previous experimental) and 3-methoxybenzylamine(1.37 g, 10.0 mmol) in isopropanol (3 mL) is stirred under N₂ at refluxfor 3 h. Evaporation of the solvent and chromatography on silica gel(5-10% EtOH/EtOAc) gives7-amino-4-(3-methoxybenzylamino)pyrido[4,3-d]pyrimidine (153 mg, 77%) asa white solid. ¹ H NMR (DMSO) δ 9.11 (1H, s), 8.83 (1H, t, J=5.7 Hz),8.26 (1H, s), 7.24 (1H, dt, J_(d) =0.8 Hz, J_(t) =8.1 Hz), 6.92 (2H, m),6.81 (1H, dt, J_(d) =8.2 Hz, J_(t) =1.2 Hz), 6.46 (2H, brs), 6.37 (1H,s), 4.71 (2H, d, J=5.8 Hz), 3.73 (3H, s).

EXAMPLE 46 7-Amino-4-(4-chlorobenzylamino)pyrido[4,3-d]pyrimidinemesylate

The free base (56 mg, 0.20 mmol) (prepared from 2,4-diamino,5-cyanopyridinium acetate, formic acid and 4-chlorobenzylamine at 200°C. as described in a previous example is precipitated from acetonesolution with methanesulfonic acid (105 μL, 0.23 mmol) to give apolymesylate salt. ¹ H NMR (DMSO) δ 10.59 (1H, t, J=5.6 Hz), 9.24 (1H,s), 8.69 (1H, s), 7.42 (4H, s), 6.42 (1H, s), 5.8 (˜6H, vbrs), 4.89 (2H,d, J=5.8 Hz), 2.41 (˜7.5H, s).

EXAMPLE 47 7-Amino-4-(2-bromobenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (225 mg, 1.17mmol) (described in a previous experimental) and 2-bromobenzylamine(0.84 g, 4.52 mmol) is stirred under N₂ at 140° C. for 1 h, and theresulting product chromatographed on silica gel (1-5% EtOH/EtOAc) togive 7-amino-4-(2-bromobenzylamino)pyrido[4,3-d]pyrimidine (175 mg, 45%)as a light brown solid. ¹ H NMR (DMSO) δ 9.16 (1H, s), 8.85 (1H, t,J=5.7 Hz), 8.24 (1H, s), 7.64 (1H, d, J=7.8 Hz), 7.34 (1H, dd, J=7.7,7.1 Hz), 7.31 (1H, dd, J=7.7, 2.4 Hz), 7.21 (1H, ddd, J=7.8, 6.9, 2.4Hz), 6.50 (2H, brs), 6.39 (1H, s), 4.74 (2H, d, J=5.7 Hz).

EXAMPLE 48 7-Amino-4-(3-bromobenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (228 mg, 1.19mmol) (described in a previous experimental) and 3-bromobenzylamine(0.84 g, 4.52 mmol) is stirred under N₂ at 140° C. for 1 h. Theresulting product is chromatographed on silica gel (2-10% EtOH/EtOAc) togive 7-amino-4-[(3-bromophenyl)methylamino]pyrido[4,3-d]pyrimidine (203mg, 52%) as a light brown solid. ¹ H NMR (DMSO) δ 9.09 (1H, s), 8.86(1H, t, J=5.8 Hz), 8.26 (1H, s), 7.54 (1H, s), 7.44 (1H, d, J=7.8 Hz,),7.36 (1H, d, J=7.6 Hz), 7.29 (1H, t, J=7.7 Hz), 6.48 (2H, s), 6.37 (1H,s), 4.73 (2H, d, J=5.8 Hz).

EXAMPLE 49 7-Amino-4-(4-bromobenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (234 mg, 1.22mmol) (described in a previous experimental) and 4-bromobenzylamine(0.84 g, 4.52 mmol) is stirred under N₂ at 140° C. for 1 h, and theresulting product chromatographed on silica gel (10% EtOH/EtOAc) to give7-amino-4-(4-bromobenzylamino)pyrido[4,3-d]pyrimidine (192 mg, 48%) as acream solid. ¹ H NMR (DMSO) δ 9.09 (1H, s), 8.87 (1H, t, J=5.7 Hz), 8.25(1H, s), 7.51 (2H, d, J=8.3 Hz), 7.31 (2H, d, J=8.3 Hz), 6.46 (2H, brs),6.37 (1H, s), 4.70 (2H, d, J=5.8 Hz).

EXAMPLE 507-Amino-4-(2-trifluoromethylbenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (225 mg, 1.17mmol) and 2-(trifluoromethyl)benzylamine (0.90 mL, 6.42 mmol) is stirredunder N₂ at 150° C. for 1 h. The resulting product is chromatographed onsilica gel (5% EtOH/EtOAc) to give 7-amino-4-(2-trifluoromethylbenzyl)aminopyrido[4,3-d]pyrimidine (0.22 g, 59%) as a white solid. ¹ H NMR(DMSO) δ 9.16 (1H, s), 8.88 (1H, t, J=5.7 Hz), 8.23 (1H, s), 7.75 (1H,d, J=7.7 Hz), 7.62 (1H, t, J=7.5 Hz), 7.50 (1H, d, J=7.4 Hz), 7.47 (1H,t, J=7.6 Hz), 6.51 (2H, brs), 6.39 (1H, s), 4.92 (2H, d, J=5.5 Hz).

EXAMPLE 517-Amino-4-(3-trifluoromethylbenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (225 mg, 1.17mmole) and 3-(trifluoromethyl)benzylamine (0.63 mL, 4.40 mmole) isstirred under N₂ at 140° C. for 1 h. The resulting product ischromatographed on silica gel (3-5% EtOH/EtOAc) to give7-amino-4-[(3-trifluoromethylphenyl)methylamino]pyrido[4,3-d]pyrimidine(0.24 g, 63%) as a light brown solid. ¹ H NMR (DMSO) δ 9.10 (1H, s),8.92 (1H, t, J=5.7 Hz), 8.26 (1H, s), 7.71 (1H, s), 7.66 (1H, d, J=7.4Hz), 7.62 (1H, d, J=7.8 Hz), 7.57 (1H, t, J=7.6 Hz), 6.49 (2H, brs),6.38 (1H, s), 4.82 (2H, d, J=5.8 Hz).

EXAMPLE 527-Amino-4-(4-trifluoromethylbenzylamino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (225 mg, 1.17mmol) and 4-(trifluoromethyl)benzylamine (0.63 mL, 4.42 mmol) is stirredunder N₂ at 140° C. for 1 h. The resulting product is chromatographed onalumina (5-10% EtOH/CHCl₃) then silica gel (2-10% EtOH/EtOAc) to give7-amino-4-[(4-trifluoromethylphenyl)methylamino]pyrido[4,3-d]pyrimidine(0.21 g, 56%) as a light brown solid. ¹ H NMR (DMSO) δ 9.12 (1H, s),8.94 (1H, t, J=5.8 Hz), 8.24 (1H, s), 7.69 (2H, d, J=8.1 Hz), 7.56 (2H,d, J=8.1 Hz), 6.48 (2H, brs), 6.38 (1H, s), 4.82 (2H, d, J=5.8 Hz).

EXAMPLE 53 7-Amino-4-(thien-2-ylmethylamino)[4,3-d]pyrimidine dimesylate

The compound is obtained from 2,4-diamino, 5-cyanopyridinium acetate(190 mg, 0.98 mmol), formic acid (0.23 g, 4.4 mmol) andthienylmethylamine (1.07 ml, 10 mmol) as described in a previousexperimental. The crude product is converted into a dimesylate salt asdescribed previously and recrystallized from Pr^(i) OH to give7-amino-4-(thien-2-ylmethylamino)pyrido[4,3-d]pyrimidine dimesylate in19% yield. 1H NMR (DMSO) δ 10.67 (1H, t, J=5.8 Hz), 9.21 (1H, s), 8.77(1H, s), 7.48 (1H, dd, J=5.1, 1.2 Hz) 7.16 (1H, dd, J=3.4, 0.7 Hz), 7.02(1H, dd, J=4.8, 3.4 Hz), 6.42 (1H, s), 5.06 (2H, d, J=5.7 Hz), 2.41 (6H,s).

EXAMPLE 54 7-Acetylamino-4-benzylaminopyrido[4,3-d]pyrimidine

7-Acetylamino-4-methylthiopyrido[4,3-d]pyrimidine. Acetyl chloride (0.70mL, 9.84 mmol) is added to a solution of7-amino-4-methylthiopyrido[4,3-d]pyrimidine (0.20 g, 1.04 mmol)(described in a previous experimental) and Et₃ N (1.51 mL, 10.8 mmol) inTHF at 0° C., and then the mixture is stirred at 20° C. for 4 h. Water(50 mL) was added, then the solution was extracted with EtOAc (3×50 mL).Evaporation and chromatography on alumina (1% EtOH/CHCl₃) yields7-acetylamino-4-methylthiopyrido[4,3-d]pyrimidine (0.12 g, 49%) as ayellow solid. ¹ H NMR (DMSO) δ 11.05 (1H, s), 9.30 (1H, s), 9.02 (1H,s), 8.38 (1H, s), 2.71 (3H, s), 2.18 (3H, s)

7-Acetylamino-4-benzylaminopyrido[4,3-d]pyrimidine. A mixture of7-acetylamino-4-methylthiopyrido[4,3-d]pyrimidine (0.40 g, 1.71 mmol)and benzylamine (1.0 mL, 9.15 mmol) is stirred under N₂ at 140° C. for 1h, and the resulting product is chromatographed on silica gel (EtOAc) togive 7-acetylamino-4-benzylaminopyrido[4,3-d]pyrimidine (0.31 g, 62%) asa white solid. ¹ H NMR (DMSO) δ 10.79 (1H, s), 9.42 (1H, s), 9.23 (1H,t, J=5.8 Hz), 8.49 (1H, s), 8.18 (1H, s), 7.39 (1H, dt, J_(d) =6.9 Hz,J_(t) =1.7 Hz), 7.34 (1H, tt, J=7.3, 1.7 Hz), 7.25 (1H, tt, J=7.1, 1.7Hz), 4.80 (2H, d, J=5.8 Hz), 2.15 (3H, s).

EXAMPLE 55 4-Anilinopyrido[3,4-d]pyrimidine

4-Carboxamidonicotinic acid. 3,4-Pyridine dicarboxylic anhydride (8.3 g,55.6 mmol) is added to conc NH₄ OH (12 mL) in H₂ O (60 mL) stirred at 0°C. over 5 min. Upon addition a paste forms which is stirred for 1 h atroom temperature. The white paste is sparged with N₂ for 30 min anddiluted with H₂ O (10 mL) to form a clear solution. Then SO₂ is bubbledthrough the solution for 15 min reducing its pH to 2. Upon cooling theresulting solid is filtered, rinsed with H₂ O, and oven dried to yield4-carboxamidonicotinic acid (7 g, 76%) as a white solid. ¹ H NMR (DMSO)δ 8.93 (1H, s), 8.76 (1H, d, J=5.0 Hz), 8.08 (1H, s), 7.62 (1H, s), 7.45(1H, d, J=5.0 Hz).

Isoquinolinic imide. 4-Carboxamidonicotinic acid (280 mg, 1.68 mmol) isheated neat at 200° C. for 5 h to yield isoquinolinic imide (177.2 mg,71%) as a white solid. ¹ H NMR (DMSO) δ 11.68 (1H, s), 9.12-9.03 (2H,m), 7.80 (1H, d, J=5.1 Hz).

3-Amino isonicotinic acid. Bromine (1.71 g) is added to 10% KOH (30 mL)on ice. The resulting solution is added to finely ground isoquinolinicimide (1.46 g, 9.86 mmol). Upon addition the mixture begins to foam.When all of the solid is dissolved up aqueous KOH (15%, 7 mL) is addedand the mixture is heated to 80° C. for 1 min then cooled. The mixtureis neutralized with SO₂, and cooled to 0° C. until precipitation occurs.The solid is collected by suction filtration and washed with H₂ O, anddried in a vacuum oven to yield of 3-amino isonicotinic acid (485 mg,36%) as a white solid. ¹ H NMR (DMSO) δ 9.5-8.8 (2H, brs), 8.20 (1H, s),7.70 (1H, d, J=5 Hz), 7.46 (1H, d, J=5 Hz).

3H-Pyrido[3,4-d]pyrimid-4-one. A mixture of 3-amino isonicotinic acid(485 mg, 3.51 mmol) in formamide (3 mL) is heated to 160° C. for 12 h.Upon cooling, the resulting solid is filtered and washed with H₂ O anddried in a vacuum oven to yield 3H-pyrido[3,4-d]pyrimid-4-one (373 mg,72%). ¹ H NMR (DMSO) δ 12.60 (1H, brs), 9.06 (1H, s), 8.68 (1H, d, J=5.3Hz), 8.23 (1H, s), 7.96 (1H, d, J=5.1 Hz).

4-Thiopyrido[3,4-d]pyrimidine. Phosphorous pentasulfide (1.25 g, 2.74mmol) is added to a solution of 3H-pyrido[3,4-d]pyrimid-4-one (366 mg,2.49 mmol) in pyridine (4 mL). The mixture is refluxed for 4 h under N₂.The resulting black tar is dissolved in H₂ O, and a solid forms. Thesolid is filtered and washed with H₂ O and dried in a vacuum oven toyield 4-thiopyrido[3,4-d]pyrimidine (369.8 mg, 91%) as a yellow solid.1H NMR (DMSO) δ 14.48 (1H, brs), 9.13 (1H, s), 8.70 (1H, d, J=5.4 Hz),8.29 (1H, s), 8.27 (1H, d, J=5.4 Hz).

4-Methylthiopyrido[3,4-d]pyrimidine. A mixture of4-thiopyrido[3,4-d]pyrimidine (369.8 mg, 2.26 mmol), triethylamine (0.6mL, 4.5 mmol), DMSO (2 mL), and iodomethane (0.24 mL, 3.96 mmol) isstirred under N2 at 25° C. for 12 h. The mixture is poured into H₂ O andthe resulting solid is filtered and dried in a vacuum oven to yield4-methylthiopyrido[3,4-d]pyrimidine (222 mg, 55%) as a brown solid. ¹ HNMR (DMSO) δ 9.51 (1H, s), 9.18 (1H, s), 8.79 (1H, d, J=8 Hz), 7.97 (1H,d, J=8 Hz).

4-Anilinopyrido[3,4-d]pyrimidine. A mixture of4-methylthiopyrido[3,4-d]pyrimidine (75 mg, 0.42 mmol), and aniline (1mL) is heated to 100° C. under N₂ for 2 h. The reaction mixture is thenchromatographed on silica using MPLC and eluting with a gradient system(CHCl₃ to 5% MeOH in CHCl₃). The fractions are concentrated underreduced pressure, and the resulting solid is recrystallized from Et₂ Oto yield 4-anilinopyrido[3,4-d]pyrimidine (21.2 mg, 23%) as a yellowsolid. ¹ H NMR (DMSO) δ 10.09 (1H, s), 9.18 (1H, s), 8.74 (1H, d, J=5.3Hz), 8.46 (1H, d, J=5.8 Hz), 7.89 (2H, d, J=8.5 Hz), 7.45 (2H, t, J=7.9Hz), 7.21 (1H, t, J=7.4 Hz).

EXAMPLE 56 4-(3-Bromoanilino)pyrido[3,4-d]pyrimidine

A mixture of 4-methylthiopyrido[3,4-d]pyrimidine (75 mg, 0.42 mmol) (seeprevious experimental), and 3-bromoaniline (1 mL) is heated to 100° C.under N₂ for 2 h. The reaction mixture is then chromatographed on silicausing MPLC and eluting with a gradient system (CHCl₃ to 5% MeOH inCHCl₃). The fractions are concentrated under reduced pressure, and theresulting solid is recrystallized from Et₂ O to yield4-(3-bromoanilino)pyrido[3,4-d]pyrimidine (66 mg, 52.7%) as a lightbrown solid. ¹ H NMR (DMSO) δ 10.15 (1H, s), 9.21 (1H, s), 8.80 (1H, s),8.76 (1H, d, J=5.8 Hz), 8.44 (1H, d, J=5.6 Hz), 8.25 (1H, s), 7.93 (1H,d, J=7.7 Hz), 7.45-7.37 (2H, m).

EXAMPLE 57 4-(3-Bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine

5-[N-(tert-Butoxycarbonyl)amino]-2-fluoropyridine.5-Amino-2-fluoropyridine is prepared by hydrogenation (Pd/C) of2-fluoro-5-nitropyridine (obtained from from 2-chloro-5-nitropyridine byreaction with KF in MeCN with Ph₄ PBr [J. H. Clark and D. J. Macquarrie,Tetrahedron Lett., 1987, 28, 111-141. Reaction of the crude amine witht-Boc anhydride gives 5-[N-(tert-butoxycarbonyl)amino]-2-fluoropyridine.¹ H NMR (CDCl₃ δ 8.07 (1H, s), 8.05 (1H, m), 6.89 (1H, dd, J=9.2, 3.3Hz), 6.66 (1H, m), 1.52 (9H, s).

5-[N-(tert-Butoxycarbonyl)amino]-2-fluoropyridine-4-carboxylic acid.Reaction of 5-[N-(tert-butoxycarbonyl)amino]-2-fluoropyridine (5.3 g, 25mmol) sequentially with n-BuLi and CO₂ as described in the followingexample gives5-[N-(tert-butoxycarbonyl)amino]-2-fluoropyridine-4-carboxylic acid(1.60 g, 25%). ¹ H NMR (DMSO) δ 9.83 (1H, brs), 8.84 (1H, s), 7.49 (1H,d, J=2.9 Hz), 1.47 (9H, s).

5-Amino-2-fluoropyridine-4-carboxylic acid. Reaction of5-[N-(tert-butoxycarbonyl)amino]-2-fluoropyridine-4-carboxylic acid (1.0g, 3.9 mmol) with TFA as described above gives5-amino-2-fluoropyridine-4-carboxylic acid(0.46 g, 74%). ¹ H NMR (DMSO)δ 7.85 (1H, d, J=1.5 Hz), 7.23 (1H, d, J=2.5 Hz).

6-Fluoro-3H-pyrido[3,4-d]pyrimidin-4-one. Reaction of5-amino-2-fluoropyridine-4-carboxylic acid with formamide at 140° C. asabove gave 6-fluoro-3H-pyrido[3,4-d]pyrimidin-4-one (˜20%). ¹ H NMR(DMSO) δ 12.48 (1H, m), 8.74 (1H, s), 8.16 (1H, s), 7.63 (1H, d, J=3Hz).

4-(3-Bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine. Reaction of6-fluoro-3H-pyrido[3,4-d]pyrimidin-4-one (0.60 g, 3.6 mmol) with POCl₃,followed by reaction of the crude 4,6-dihalo compound with3-bromoaniline gives 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine(0.73 g, 63%). ¹ H NMR (DMSO) δ 10.09 (1H, brs), 8.96 (1H, s), 8.75 (1H,s), 8.25 (2H, m), 7.90 (1H, brd, J=6.5 Hz), 7.44-7.34 (2H, m).

EXAMPLE 58 4-(3-Bromoanilino)-6-chloropyrido[3,4-d]pyrimidine

5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine. A mixture of5-amino-2-chloropyridine (12.86 g, 0.1 mol), di-tert-butyldicarbonate(24.0 g, 0.11 mol) and Et₃ N (12.1 g, 1.12 mol) in CH₂ Cl₂ (150 mL) isheated under reflux for 12 h, cooled, and the precipitate is filteredoff. The organic layer is washed with water, dried (CaCl₂) and filteredthrough a short column of alumina. Removal of the solvent gives5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine (11.9 g, 52%). ¹ H NMR(CDCl₃) δ 8.31 (1H, d, J=2.9 Hz), 7.94 (1H, dd, J=8.6, 2.6 Hz), 7.24(1H, d, J=8.7 Hz), 7.15 (1H, m), 1.51 (9H, s).

5-[N-(tert-Butoxycarbonyl)amino]-2-chloropyridine-4-carboxylic acid. Asolution of 5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine (22.87 g,0.1 mol) and TMEDA (47 mL, 0.31 mol) in dry Et₂ O (600 mL) is cooled to-78° C., and n-BuLi (10M in hexanes, 30 mL, 0.3 mol) is added dropwise.The solution is allowed to warm to -10° C. and is then kept at thattemperature for 2 h, before being recooled to -78° C. Dry CO₂ is thenbubbled in, and the resulting mixture is allowed to warm to 20° C.,before being quenched with water (300 mL) containing a small amount ofNH₄ OH. The resulting aqueous layer is washed with EtOAc, then acidifiedslowly with dilute HCl to precipitate5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine-4-carboxylic acid(15.5 g, 57%). ¹ H NMR (DMSO) δ 10.00 (1H, s), 9.13 (1H, s), 7.74 (1H,s), 1.47 (9H, s).

5-Amino-2-chloropyridine-4-carboxylic acid. A stirred suspension of5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine-4-carboxylic acid(1.91 g, 7 mmol) in CH₂ Cl₂ (200 mL) is treated slowly withtrifluoroacetic acid until homogeneous (ca. 12 mL). The solution isstirred overnight and extracted with dilute NH₄ OH, and the aqueouslayer is then acidified with dilute HCl to gave a precipitate of5-amino-2-chloropyridine-4-carboxylic acid (1.05 g, 87% yield). ¹ H NMR(DMSO) δ 9.01 (2H, m), 8.03 (1H, s), 7.48 (1H, s).

6-Chloro-3H-pyrido[3,4-d]pyrimidin-4-one. A solution of5-amino-2-chloropyridine-4-carboxylic acid (8.1 g, 4.7 mmol) informamide (100 mL) is stirred at 140° C. for 12 h. Dilution of thecooled mixture with water gives a precipitate of6-chloro-3H-pyrido[3,4-d]pyrimidin-4-one (7.3 g, 86% yield). ¹ H NMR(DMSO) δ 12.73 (1H, m), 8.90 (1H, d, J=0.7 Hz), 8.23 (1H, s), 7.97 (1H,d, J=0.7 Hz).

4,6-Dichloropyrido[3,4-d]pyrimidine. A stirred suspension of6-chloropyrido[3,4-d]pyrimidin-4-one (1.82 g, 10 mmol) in POCl₃ (10 mL)is heated under reflux until dissolved (ca. 2 h) and for a further 30min. Excess reagent is removed under reduced pressure, and the residueis treated with a mixture of CH₂ Cl₂ and ice-cold aqueous Na₂ CO₃. Theresulting organic layer is dried (Na₂ SO₄) and evaporated to give aquantitative yield of crude, unstable,4,6-dichloropyrido[3,4-d]pyrimidine, which is used directly in the nextstep. ¹ H NMR (CDCl₃) δ 9.38 (1H, d, J=0.5 Hz), 9.19 (1H, s), 8.09 (1H,d, J=0.5 Hz).

4-(3-Bromoanilino)-6-chloropyrido[3,4-d]pyrimidine. A mixture of theabove crude dichloropyrimidine and 3-bromoaniline (3.8 g, 22 mmol) isdissolved in i-PrOH (100 mL). One drop of conc. HCl is added to initiatethe reaction, and the mixture is then heated under reflux for 30 min,cooled, and diluted with water to precipitate4-(3-bromoanilino)-6-chloropyrido[3,4-d]pyrimidine (1.26 g, 38% yield).¹ H NMR (DMSO) δ 10.12 (1H, s), 9.03 (1H, s), 8.77 (1H, s), 8.63 (1H,s), 8.21 (1H, s), 7.89 (1H, d, J=8.1 Hz), 7.43-7.32 (2H, m).

EXAMPLE 59 4-(3-Bromoanilino)-6-methoxypyrido[3,4-d]pyrimidine

Treatment of 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine (see aprevious experimental) at 100° C. in a pressure vessel with sodiummethoxide in methanol gives4-(3-bromoanilino)-6-methoxypyrido[3,4-d]pyrimidine. ¹ H NMR (DMSO) δ9.93 (1H, s), 8.94 (1H, s), 8.61 (1H, s), 8.26 (1H, brs), 7.94 (1H, brd,J=7.6 Hz), 7.88 (1H, s), 7.43-7.32 (2H, m), 4.01 (3H, s).

EXAMPLE 60 4-(3-Bromoanilino)-6-methylaminopyrido[3,4-d]pyrimidine

Treatment of 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine (0.20 g,0.63 mmol) (see a previous experimental) at 100° C. in a pressure vesselwith methylamine in ethanol followed by chromatography on alumina (CH₂Cl₂ /MeOH, 99:1) gives4-(3-bromoanilino)-6-methylaminopyrido[3,4-d]pyrimidine (0.07 g, 34%). ¹H NMR (DMSO) δ 9.69 (1H, s), 8.75 (1H, s), 8.41 (1H, s), 8.21 (1H, brs),7.93 (1H, brd, J=7.6 Hz), 7.41-7.28 (2H, m), 7.06 (1H, s), 6.82 (1H, q,J=5.0 Hz), 4.95 (3H, d, J=5.0 Hz).

EXAMPLE 61 4-(3-Bromoanilino)-6-dimethylaminopyrido3,4-d]pyrimidine

Treatment of 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine (see aprevious experimental) at 100° C. in a pressure vessel withdimethylamine in ethanol gives4-(3-bromoanilino)-6-dimethylaminopyrido[3,4-d]pyrimidine. ¹ H NMR(DMSO) δ 9.71 (1H, s), 8.83 (1H, s), 8.43 (1H, s), 8.21 (1H, brs), 7.94(1H, brd, J=7.5 Hz), 7.42-7.29 (2H, m), 7.26 (1H, s), 3.17 (6H, s).

EXAMPLE 62 4-(Benzylamino)pyrido[3,4-d]pyrimidine

A mixture of 4-methylthiopyrido[3,4-d]pyrimidine (74 mg, 0.41 mmol) (seea previous experimental), and benzylamine (1 mL) is heated to 100° C.for 2 h. On cooling the mixture is concentrated under reduced pressureand purified directly by preparative tlc on silica gel eluting with CH₂Cl₂, to yield 4-(benzylamino)pyrido[3,4-d]pyrimidine (21.2 mg, 20%). ¹ HNMR (DMSO) δ 9.21 (1H, t, J=5.8 Hz), 9.19 (1H, s), 8.63 (1H, d, J=5.8Hz), 8.58 (1H, s), 8.20 (1H, d, J=5.1 Hz), 7.41-7.30 (4H, m), 7.26 (1H,t, J=7.1 Hz).

EXAMPLE 63 4-(3-Bromoanilino)pyrido[2,3-d]pyrimidine

3H-pyrido[2,3-d]pyrimidin-4-one. 2-Amino nicotinic acid (15 g, 108.6mmol) in formamide (35 mL) is heated to 165-170° C. for 3.5 h. Uponcooling a solid precipitates. The solid is filtered and washed with H₂ Oand dried in a vacuum oven to give 3H-pyrido[2,3-d]pyrimidin-4-one (7.87g, 49.4%). 1H NMR (DMSO) δ 12.50 (1H, s), 8.97 (1H, dd, J=1.9, 4.5 Hz),8.53 (1H, dd, J=2.1, 7.9 Hz), 8.34 (1H, s), 7.57 (1H, dd, J=4.6, 8.0Hz).

4-Thiopyrido[2,3-d]pyrimidine. Phosphorous pentasulfide (6 g, 13.5 mmol)is added to a solution of 3H-pyrido[2,3-d]pyrimidin-4-one (2 g, 13.5mmol) in pyridine (50 mL). The mixture is refluxed for 3 h. Upon coolinga solid formed and the pyridine is decanted off. The solid is suspendedin H₂ O (20 mL) and then filtered and dried in a vacuum oven to yield4-thiopyrido[2,3-d]pyrimidine (1.72 g, 78%). ¹ H NMR (DMSO) δ 9.06 (1H,dd, J=1.9, 4.3 Hz), 8.90 (1H, dd, J=1.9, 8.2 Hz), 8.36 (1H, s), 7.65(1H, dd, J=4.3, 8.2 Hz).

4-Methylthiopyrido[2,3-d]pyrimidine. A mixture of4-thiopyrido[2,3-d]pyrimidine (100 mg, 0.76 mmol), triethylamine (154mg, 1.52 mmol), DMSO (2 mL), and iodomethane (161 mg, 1.14 mmol) isstirred for 12 h at 25° C. The mixture is poured into H₂ O and extractedwith EtOAc. The combined extracts are washed with water, saturatedbrine, and dried (MgSO₄), and the solvent is removed under reducedpressure to yield 4-methylthiopyrido[2,3-d]pyrimidine (134 mg, quant.).¹ H NMR (DMSO) δ 9.25 (1H, dd, J=1.8, 4.2 Hz), 9.17 (1H, s), 8.59 (1H,dd, J=1.9, 8.2 Hz), 7.75 (1H, dd, J=4.3, 8.2 Hz), 2.73 (3H, s).

A mixture of 4-methylthiopyrido[2,3-d]pyrimidine (157 mg, 0.89 mmol, and3-bromoaniline (1 mL) is heated to 100° C. for 2 h. On cooling aprecipitate forms which is filtered then washed with EtOH and air driedto yield 4-(3-bromoanilino)pyrido[2,3-d]pyrimidine (55.5 mg, 20%. ¹ HNMR (DMSO) δ 10.13 (1H, s), 9.11 (1H, dd, J=1.7, 4.3 Hz), 9.01 (1H, dd,J=1.7, 8.2 Hz), 8.81 (1H, s), 8.22 (1H, s), 7.90 (1H, d, J=7.7 Hz), 7.71(1H, dd, J=4.3, 8.0 Hz), 7.40 (2H, m).

EXAMPLE 64 4-(3-Bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine

2,6-Difluoronicotinic acid. 2,6-Difluoropyridine (7.89 mL, 0.087 mmol)is added dropwise under N₂ at 78° C. to a stirred solution of lithiumdiisopropylamide (59.0 mL of a 1.5N solution in cyclohexane, 0.089 mmol)in THF (250 mL). After 2 h at 78° C., a stream of dry CO₂ is passedthrough the solution and the mixture is diluted with water and washedwith EtOAc. The aqueous portion is neutralized with 3N HCl, extractedwith EtOAc and worked up to give 2,6-difluoronicotinic acid (13.4 g,97%). ¹ H NMR (DMSO) δ 8.59 (1H, dd, J=9.2, 8.2 Hz), 7.30 (1H, dd,J=8.2, 2.1 Hz), 4.03 (1H, brs).

2,6-Difluoronicotinamide. A solution 2,6-difluoronicotinic acid (7.4 g,0.046 mmol) and SOCl₂ (20 mL) in 1,2-dichloroethane (60 mL) containingDMF (1 drop) is heated under reflux for 4 h, then concentrated todryness under reduced pressure. The residue is dissolved in Et₂ O (100mL), cooled to 0° C., and treated dropwise with concentrated ammonia(10.0 mL, 0.17 mmol). After 10 min the solution is washed with aqueousNaHCO₃ and worked up to give 2,6-difluoronicotinamide (5.61 g, 76%). ¹ HNMR (CDCl₃) δ 8.70 (1H, dd, J=9.6, 8.3 Hz), 7.00 (1H, ddd, J=8.3, 2.9,1.1 Hz), 6.71, 6.55 (1H, 1H, 2 brs).

2-Amino-6-fluoronicotinamide. A solution of 2,6-difluoronicotinamide(4.68 g, 0.029 mmol) in dry formamide (30 mL) is saturated with ammoniaand allowed to stand at room temperature for 24 h. Water (50 mL) isadded and the resultant precipitate is filtered off and washed well withwater, to give 6-amino-2-fluoronicotinamide (1.41 g, 31%) mp 236-237° C.¹ H NMR (DMSO) δ 7.89 (1H, dd, J=10.4, 8.4 Hz), 7.31, 7.16 (1H, 1H, 2brs,), 6.93 (2H, brs), 6.36 (1H, dd, J=8.4, 2.4 Hz).

The filtrate and washings are combined and extracted exhaustively withEtOAc, and the extract is chromatographed on silica gel. EtOAc/petroleumether (1:1) elutes forerun, while EtOAc/petroleum ether (2:1) and thenEtOAc gives 2-amino-6-fluoronicotinamide (1.57 g, 35%), mp(EtOAc/petroleum ether) 199-200° C. [Rogers, R. B. et al., U.S. Pat. No.4,383,851, record mp 198-200° C.]. ¹ H NMR (DMSO) δ 8.13 (1H, dd,J=10.4, 8.4 Hz), 7.90, 7.30 (1H, 1H, 2 brs), 7.65 (2H, brs), 6.23 (1H,dd, J=8.4, 2.6 Hz).

A suspension of 2-amino-6-fluoronicotinamide (0.74 g, 4.77 mmol) intriethyl orthoformate (25 mL) is heated at reflux for 8 h. After coolingto room temperature the precipitate is filtered off and washed well withpetroleum ether to give 7-fluoropyrido[2,3-d]pyrimid-4(3H)-one (0.76 g,96%). ¹ H NMR (DMSO) δ 12.75 (1H, brs), 8.66 (1H, dd, J=10.4, 8.4 Hz),8.38 (1H, s), 7.33 (1H, dd, J=8.4, 2.6 Hz).

4-(3-Bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine. A suspension of7-fluoropyrido[2,3-d]pyrimid-4 (3H)-one (0.20 g, 1.21 mmol) in POCl₃ (10mL) is heated under reflux for 2 h. The volatiles are then removed underreduced pressure, and the residue is partitioned between aqueous NaHCO₃and EtOAc. The organic extract is worked up to give crude4-chloro-7-fluoropyrido[2,3-d]pyrimidine, which is used directly in thenext reaction. A solution of this product (0.20 g, 1.09 mmol) and3-bromoaniline (0.23 mL, 2.18 mmol) in propan-2-ol (1.0 mL) and THF (10mL) containing a trace of conc. HCl is stirred at 20° C. for 1 h, andthen concentrated to dryness. The residue is dissolved in EtOAc, washedwith aqueous NaHCO₃, and worked up to give an oil, which ischromatographed on silica gel. Elution with EtOAc/petroleum ether (1:5)gives 3-bromoaniline, while EtoAc/petroleum ether (1:1) elutes4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine (0.18 g, 47%), mp(MeOH) 211-213° C. ¹ H NMR (DMSO) δ 10.18 (1H, brs), 9.17 (1H, t, J=8.6Hz), 8.80 (1H, s), 8.17 (1H, t, J=1.8 Hz), 7.85 (1H, dt, J_(d) =7.6 Hz,J_(t) =1.8 Hz), 7.53 (1H, dd, J=8.6, 2.7 Hz), 7.41-7.34 (2H, m).

EXAMPLE 65 7-Amino-4-(3-bromoanilino)pyrido[2,3-d]pyrimidine

A solution of 4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine (0.20g, 0.63 mmol) in EtOH (20 mL) is saturated with ammonia and warmed at100° C. in a pressure vessel for 30 h. The solvent is removed underreduced pressure to give7-amino-4-(3-bromoanilino)pyrido[2,3-d]pyrimidine (0.18 g, 90%). ¹ H NMR(DMSO) δ 9.97 (1H, brs), 8.59 (1H, s), 8.51 (1H, d, J=9.3 Hz), 8.11 (1H,sl brs), 7.77 (1H, brd, J=6.3 Hz), 7.44 (2H, brs), 7.37-7.30 (2H, m),6.81 (1H, d, J=9.3 Hz).

EXAMPLE 66 4-(3-Bromoanilino)-7-methylaminopyrido[2,3-d]pyrimidine

A solution of 4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine (see aprevious experimental) (0.20 g, 0.63 mmol), methylamine hydrochloride(0.13 g, 1.88 mmol) and Et₃ N (0.30 mL) 2.19 mmol) in EtOH (15 mL) isheated at 100° C. in a pressure vessel for 18 h. The solvent is removedunder reduced pressure, and the residue is partitioned between EtOAc andwater. Workup of the organic layer gives4-(3-bromoanilino)-7-(methylamino)pyrido[2,3-d]pyrimidine (0.16 g, 77%).¹ H NMR (DMSO) δ 9.53 (1H, s), 8.54 (1H, s), 8.41 (1H, d, J=8.1 Hz),8.17 (1H, t, J=1.8 Hz), 7.83 (1H, dd, J=8.0, 1.9 Hz), 7.66 (1H, brs),7.32 (1H, t, J=8.0 Hz), 7.24 (1H, dd, J=8.0, 1.8 Hz), 6.77 (1H, d, J=8.1Hz), 2.92 (3H, d, J=4.8 Hz).

EXAMPLE 67 4-(3-Bromoanilino)-7-dimethylaminopyrido[2,3-d]pyrimidine

Reaction of 4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine (see aprevious experimental) (0.12 g, 0.38 mmol) with dimethylaminehydrochloride (92 mg, 1.13 mmol) and Et₃ N (0.18 mL, 1.32 mmol) in EtOH(15 mL) at 100° C. for 18 h in a pressure vessel, followed byevaporation of the solvent and workup, gives4-(3-bromoanilino)-7-(dimethylamino)pyrido[2,3-d]pyrimidine (0.11 g,84%). ¹ H NMR (DMSO) δ 9.58 (1H, brs), 8.56 (1H, d, J=9.3 Hz), 8.54 (1H,s), 8.18 (1H, t, J=1.9 Hz), 7.84 (dt, J_(d) =8.0 Hz, J_(t) =1.9 Hz),7.33 (1H, dd, J=8.1, 8.0 Hz) 7.25 (1H, dt, J_(d) =9.3 Hz, J_(t) =1.9Hz), 7.10 (1H, d, J=9.3 Hz), 3.18 (6H, s).

EXAMPLE 68 4-(3-Bromoanilino)-7-methoxypyrido[2,3-d]pyrimidine

A solution of 4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine (0.26g, 0.81 mmol) and sodium methoxide (prepared from 75 mg of sodium, 3.26mmol) in dry MeOH (15 mL) is heated at 90° C. in a pressure vessel for18 h. The mixture is poured into water and extracted with EtOAc to give4-(3-bromoanilino)-7-methoxypyrido[2,3-d]pyrimidine (0.23 g, 86%). ¹ HNMR (DMSO) δ 9.88 (1H, brs), 8.82 (1H, d, J=8.9 Hz), 8.71 (1H, s), 8.18(1H, dd, J=8.0, 1,9 Hz), 7.36 (1H, dd, J=8.1, 8.0 Hz), 7.29 (1H, ddd,J=8.1, 1.9, 1,9 Hz) 7.15 (1H, d, J=8.9 Hz), 4.01 (3H, s).

EXAMPLE 69 4-Benzylamino-7-methylaminopyrimido[4,5-d]pyrimidine

S-Ethylisothiouronium iodide. A solution of thiourea (3.80 g, 50 mmol)and iodoethane (4 mL, 50 mmol) in MeOH (100 mL) is refluxed for 24 h.The solvent is stripped under reduced pressure, and the residual lightyellow oil, is dried under vacuum, solidifying spontaneously. Thedesired compound (13.98 g) is obtained quantitatively.

4-Amino-5-cyano-2-ethylthiopyrimidine. A suspension of NaOMe (2.7 g, 50mmol) in EtOH (200 mL) is added to a mixture of S-ethylisothioureahydroiodide (11.58 g, 50 mmol), ethoxymethylidenemalononitrile (6.1 g,50 mmol) and ethanol (250 mL) at 25° C. The reaction mixture is refluxedunder N₂ for 2 h, and then the solution is concentrated on a hot plateuntil precipitation is observed. After cooling, the solid is collectedby suction filtration and is stirred in water at 25° C. Filtration andvacuum oven drying affords 4-amino-5-cyano-2-ethylthiopyrimidine (4.02g, 45%) as a brown solid. ¹ H NMR δ (DMSO) 8.45 (1H, s), 7.90 (2H, brs),3.00 (2H, q, J=7.3 Hz), 1.27 (3H, t, J=7.3 Hz).

4-Amino-2-ethylthiopyrimidine-5-carboxamide.4-Amino-5-cyano-2-ethylthiopyrimidine (4.0 g, 22.3 mmol) is added tosulfuric acid (conc., 4.3 mL) in small portions. The mixture is thenstirred under N₂ at 40° C. for 1.5 h. The reaction is quenched withice-water and NH₄ OH is used to adjust the pH to ˜9. The solid iscollected via suction filtration and dried in a vacuum oven overnight.4-Amino-2-ethylthiopyrimidine-5-carboxamide (2.58 g, 58%) is obtained asa light brown solid. 1H NMR: (DMSO) δ 8.52 (1H, s), 7.98 (2H, brs), 7.42(2H, brs), 3.04 (2H, q, J=7.3 Hz), 1.27 (3H, t, J=7.3 Hz).

4-Oxo-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine. A mixture of4-amino-2-ethylthiopyrimidine-5-carboxamide (4.66 g, 23.5 mmol) andtriethyl orthoformate (150 mL) is refluxed under N₂ for 24 h, and isthen cooled to 25° C. The brown solid is isolated by suction filtrationand dried in a vacuum oven to give4-oxo-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine (3.54 g, 72%). ¹ H NMR:(DMSO) δ 12.80 (1H, s), 9.20 (1H, s), 8.45 (1H, s), 3.18 (2H, q, J=7.4Hz), 1.35 (3H, t, J=7.4 Hz).

4-Thiono-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine. A mixture of4-oxo-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine (1.33 g, 6.7 mmol), P₂ S₅(1.48 g, 6.6 mmol) and pyridine (15 mL) is refluxed under N₂ for 3 h.The pyridine is then stripped under reduced pressure, and the residue isdissolved in NaOH solution (0.5M, 75 mL) and boiled with charcoal. Afterfiltration, the filtrate is neutralized with acetic acid to generate agold brown solid. Buchner filtration and drying in a vacuum oven affords4-thiono-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine (1.42 g, 95%). ¹ H NMR(DMSO) δ 9.47 (1H, s), 8.46 (1H, s), 3.20 (2H, q, J=7.3 Hz), 1.35 (3H,t, J=7.3 Hz).

7-Ethylthio-4-methylthiopyrimido[4,5-d]pyrimidine. The same proceduredescribed for 7-amino-4-methylthiopyrido[4,3-d]pyrimidine in Example 21is used. ¹ H NMR (DMSO) δ 9.52 (1H, s), 9.15 (1H, s), 3.23 (2H, q, J=7.3Hz), 2.72 (3H, s), 1.38 (3H, t, J=7.3 Hz).

4-Benzylamino-7-ethylthiopyrimido[4,5-d]pyrimidine. The same proceduredescribed for 7-amino-4-anilinopyrido[4,3-d]pyrimidine in example 21 isused.

4-Benzylamino-7-methylaminopyrimido[4,5-d]pyrimidine.4-Benzylamino-7-ethylthiopyrimido[4,5-d]pyrimidine in EtOH containingexcess methylamine is heated to 150° C. in a stainless steel bomb for 5h. The solid is filtered off and dried to give4-benzylamino-7-methylaminopyrimido[4,5-d]pyrimidine.

EXAMPLE 70 4-Benzylamino-7-hydrazinopyrimido[4,5-d]pyrimidine

4-Benzylamino-7-ethylthio pyrimido[4,5-d]pyrimidine in EtOH containingexcess hydrazine is heated to 150° C. in a stainless steel bomb for 5 h.The solid is filtered off and dried to give4-benzylamino-7-hydrazinopyrimido[4,5-d]pyrimidine. ##STR14##

EXAMPLE 71 4-(3-Bromoanilino)thieno[3,2-d]pyrimidine hydrochloride

3H-Thieno[3,2-d]pyrimid-4-one. A mixture of methyl3-aminothiophene-2-carboxylate (1 g, 6.3 mmol) and formamide (2 g) isheated at 240° C. for 10 min. Upon cooling a precipitate appeared. It isdissolved in EtOH and filtered. The filtrate is concentrated underreduced pressure and the residue is purified by silica gelchromatography eluting with 10% MeOH in CH₂ Cl₂ to yield3H-thieno[3,2-d]pyrimid-4-one (249 mg, 26%) as a solid. ¹ H NMR (DMSO) δ12.61 (1H, brs), 8.20 (1H, s), 8.17 (1H, d, J=5 Hz), 7.42 (1H, d, J=5Hz).

4-Chlorothieno[3,2-d]pyrimidine. To a solution of DMF (170.3 μL, 2.2mmol) and dichloroethane (1.2 mL) at 0° C. under N₂, oxalyl chloride(279.2 mg, 3.2 mmol) is added slowly and stirred for 10 min.3H-thieno[3,2-d]pyrimid-4-one (152.2 mg, 1.0 mmol) is added and refluxedfor 5 h. The reaction mixture is poured into water and extracted withCH₂ Cl₂. The organic layer is stripped under reduced pressure to yield4-chlorothieno[3,2-d]pyrimidine (140 mg, 82%) as a yellow solid. ¹ H NMR(DMSO) δ 9.05 (1H, s), 8.62 (1H, d, J=5 Hz), 7.79 (1H, d, J=5 Hz).

4-(3-Bromoanilino)thieno[3,2-d]pyrimidine hydrochloride. A mixture of4-chlorothieno[3,2-d]pyrimidine (135 mg, 0.79 mmol) and 3-bromoaniline(95 μL, 0.89 mmol) in 2-methoxyethanol (2 mL) is heated to 79° C. for 30min. The resulting precipitate is filtered and washed with CH₂ Cl₂ toyield 4-(3-bromoanilino)thieno[3,2-d]pyrimidine hydrochloride (195.5 mg,72%) as a light yellow solid. ¹ H NMR (DMSO) δ 11.33 (1H, s), 8.94 (1H,s), 8.23 (1H, s), 8.53 (1H, d, J=5.3 Hz), 8.07 (1H, s), 7.77 (1H, d,J=7.9 Hz), 7.6 (1H, d, J=5.3 Hz), 7.48 (2H, m).

EXAMPLE 72 4-Benzylaminothieno[3,2-d]pyrimidine

As described in the previous experiment 4-chlorothieno[3,2-d]pyrimidine(100 mg, 0.586 mmol) and benzylamine (710 μL, 0.645 mmol) in2-methoxyethanol (2 mL) yields 4-benzylaminothieno[3,2-d]pyrimidine (37mg, 26%). ¹ H NMR (DMSO) δ 8.42 (1H, s), 8.12 (1H, d, J=5.5 Hz), 7.39(1H, d, J=5.3 Hz), 7.40-7.30 (4H, m), 7.24 (1H, t, J=6.8 Hz).

EXAMPLE 73 4-(3-Bromoanilino)thieno[2,3-d]pyrimidine

Methyl 2-aminothiophene-3-carboxylate. A mixture of methyl cyanoacetate(3.25 g, 32.3 mmol), 1,4 dithiane-2,5 diol (5 g, 32.8 mmol),triethylamine (1 mL, 7.71 mmol) in EtOH (50 mL) is stirred at 40° C. for1 h. The cooled solution is eluted through a silica plug with CH₂ Cl₂.The filtrate is stripped to dryness to give crude methyl2-aminothiophene-3-carboxylate which is carried on to the next reaction.¹ H NMR (DMSO) δ 7.26 (1H, s), 6.82 (1H, d, J=5.8 Hz), 6.28 (1H, d,J=5.8 Hz), 3.69 (3H, s).

3H-Thieno[2,3-d]pyrimid-4-one. A solution of methyl2-aminothiophene-3-carboxylate (602.1 mg, 3.83 mmol) in formamide (5 mL)is heated at 200° C. for 12 h. The resulting tar is dissolved in CH₂ Cl₂(10 mL) then placed on a silica plug and eluted with 10% MeOH in CH₂Cl₂. The filtrate is stripped under reduce pressure and the resultingsolid is washed with EtOH to yield 3H-thieno[2,3-d]pyrimid-4-one (231.4mg, 40%) as an orange solid. ¹ H NMR (DMSO) δ 12.50 (1H, brs), 8.13 (1H,s), 7.60 (1H, d, J=5.8 Hz), 7.41 (1H, d, J=6.0 Hz).

4-Chlorothieno[2,3-d]pyrimidine. To a solution of DMF (90 μL) and CH₂Cl₂ (2 mL) at 0° C. under N₂, oxalyl chloride (148 mg, 1.2 mmol) isadded slowly and stirred for 10 min. 3H-Thieno[2,3-d]pyrimid-4-one (81mg, 0.52 mmol) is added as a solid to the solution and warmed with aheat gun until the solid dissolves. The reaction is stirred at 25° C.for 12 h under N₂. The reaction mixture is poured into water andextracted with CH₂ Cl₂. The phases are separated and the organic layeris dried (Na₂ SO₄) and stripped under reduced pressure to yield4-chlorothieno[2,3-d]pyrimidine (87.6 mg, 97%) as a solid. ¹ H NMR(DMSO) δ 8.96 (1H, s), 8.17 (1H, d, J=6.0 Hz), 7.62 (1H, d, J=6.0 Hz).

4-(3-Bromoanilino)thieno[2,3-d]pyrimidine hydrochloride. A mixture of4-chlorothieno[2,3-d]pyrimidine (135 mg, 0.79 mmol) and 3-bromoaniline(95 μL, 0.89 mmol) in 2-methoxyethanol (2 mL) is heated to 79° C. for 30min with stirring. The resulting solid is filtered and washed with CH₂Cl₂ to yield 4-(3-bromoanilino)thieno[2,3-d]pyrimidine hydrochloride(197 mg, 73%). ¹ H NMR (DMSO) δ 9.99 (1H, s), 8.60 (1H, s), 8.23 (1H,s), 7.98 (1H, d, J=6.0 Hz), 7.88 (1H, d, J=8.0 Hz), 7.79 (1H, d, J=6.0Hz), 7.37 (1H, t, J=8.0 Hz), 7.30 (1H, d, J=8.0 Hz).

EXAMPLE 74 4-Benzylaminopyrrolo[2,3-d]pyrimidine

4-Benzylaminopyrrolo[2,3-d]pyrimidine is prepared as describedpreviously. G. H. Hitchings, K. W. Ledig and R. A. West, U.S. Pat. No.3,037,980, 1962; Chemical Abstracts 1962, 57, 15130c.

EXAMPLE 75 N⁶ -(3-Bromophenyl)adenine

A mixture of 6-chloropurine (1.0 g, 6.47 mmol), 3-bromoaniline (0.78 mL,7.12 mmol), and conc HCl (4 drops) in isopropanol (10 mL) is stirred at80° C. for 5 h. Upon cooling, it precipitates. The solid is filtered andwashed with isopropanol and air dried to yield N⁶-(3-bromophenyl)adenine (1.93 g, 91%) as a light yellow solid. ¹ H NMR(DMSO) δ 11.38 (1H, s), 8.78 (1H, s), 8.75 (1H, s), 7.90 (1H, d, J=8.0Hz), 7.38-7.34 (2H, m). ##STR15##

EXAMPLE 76 N⁶ -Benzyladenine

N⁶ -Benzyladenine is available commercially from the Aldrich ChemicalCompany, 1001 West Saint Paul Avenue, Milwaukee, Wis. 53233.

EXAMPLE 77 7-Amino-4-(3-methylanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (217 mg, 1.13mmol) and m-toluidine (1.50 g, 14.0 mmol) is stirred at 155° C. for 30min. The resulting product is chromatographed over silica gel (5%MeOH/CH₂ Cl₂) to give 7-amino-4-(3-methylanilino)pyrido[4,3-d]pyrimidine(190 mg, 67%) as a pale yellow solid. ¹ H NMR (DMSO) δ 9.81 (1H, brs),9.34 (1H, s), 8.38 (1H, s), 7.60 (2H, s), 7.26 (1H, dd, J=8.5, 7.6 Hz),6.95 (1H, d, J=7.4 Hz), 6.63 (2H, brs), 6.44 (1H, s), 2.33 (3H, s).

EXAMPLE 78 7-Amino-4-(4-methoxyanilino)pyrido[4,3-d]pyrimidine

A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (129 mg, 0.62mmol) and 4-methoxyaniline ((0.15 g, 1.2 mmol) was in ethanol (5 mL) washeated at 40° C. for 16 h, and then reflux for 3 h. The reaction mixturewas cooled to 0° C. overnignt, and the solid was colected by vacuumfiltration and recrystalized from isopropanol to give7-amino-4-(4-methoxyanilino)pyrido[4,3-d]pyrimidine (42 mg, 25%) as ayellow solid. ¹ H NMR (DMSO) δ 10.00 (1H, brs), 9.31 (1H, s), 8.35 (1H,s), 7.62 (2H, d, J=9.2 Hz), 6.96 (2H, d, J=9.2 Hz), 6.70 (2H, slbrs),6.41 (1H, s), 3.77 (3H, s).

EXAMPLE 79 4-(3-Bromoanilino)-6-(piperidin-1-yl)pyrido[3,4-d]pyrimidine

Treatment of 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine (see aprevious experimental) at 100° C. in a pressure vessel with piperidinein ethanol gives4-(3-bromoanilino)-6-dimethylaminopyrido[3,4-d]pyrimidine.

The pharmaceutical compositions of the invention can take any of a widevariety of oral and parenteral dosage forms. The dosage forms compriseas the active components an inhibitor as defined previously.

For preparing pharmaceutical compositions, one uses inert,pharmaceutically acceptable carriers that can be either solid or liquid.Solid form preparations include powders, tablets, dispersible granules,capsules, cachets, and suppositories. A solid carrier can be one or moresubstances which may also act as dilutents, flavoring agents,solubilizers, lubricants, suspending agents, binders, or tabletdisintegrating agents; it can also be an encapsulating material. Inpowders, the carrier is a finely divided solid which is in admixturewith the finely divided active compounds. In the tablet, the activecompounds are mixed with carrier having the necessary binding propertiesin suitable proportions and compacted in the shape and size desired. Thepowders and tablets preferably contain from 5% or 10% to about 70% ofactive ingredients. Suitable solid carriers are magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, alow melting wax, cocoa butter, and the like. The term "preparation" isintended to include the formulation of the active compounds withencapsulating materials as carrier, providing a capsule in which theactive components (with or without other carriers) are surrounded bycarrier, which are thus in association with it. Similarly, cachets areincluded. Tablets, powders, cachets, and capsules can be used as soliddosage forms suitable for oral administration.

Liquid form preparations include solutions, suspensions, and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection. Liquid preparations can also be formulated insolution in aqueous polyethylene glycol solution. Aqueous solutionssuitable for oral use can be prepared by dissolving the active componentin water and adding suitable colorants, flavors, stabilizing, andthickening agents as desired. Aqueous suspensions suitable for oral usecan be made by dispersing the finely divided active components in waterwith viscous material, i.e., natural or synthetic gums, resins, methylcellulose, sodium carboxymethyl cellulose, and other well-knownsuspending agents.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation may be subdivided into unit doses containingappropriate quantities of inhibitor and other anti-cancer materialsindividually or as a combination, i.e., in a mixture. The unit dosageform can be a packaged preparation, the package containing discretequantities of preparation, for example, packeted tablets, capsules, andpowders in vials or ampoules. The unit dosage form can also be acapsule, cachet, or tablet itself or it can be the appropriate number ofany of these in packaged form. Additionally, the unit dosage form may bea dividable form having an inhibitor in one part and other anti-cancermaterials in the other part, such as, a dividable capsule, a dividablepackage, or a two-part ampoule, vial or the like.

The quantity of an inhibitor in unit dosages of preparation may bevaried or adjusted from about 0.01 mg/kg to 100.0 mg/kg, preferably 0.03mg/kg to less than 1.0 mg/kg of inhibitor.

The pharmaceutical compositions preferably are constituted so that theycan be administered parenterally or orally. Solutions of the activecompounds as free bases and free acids or pharmaceutically acceptablesalts can be prepared in water suitable mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of the microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion, and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, paragens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferred to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions of agents delaying absorption, for example,gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients, into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of the sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze-drying technique whichyields a powder of active ingredients plus an additional desiredingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuitable as unitary dosages for the mammalian subjects to be treated;each unit containing a predetermined quantity of active materialscalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the noveldosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active materials andthe particular therapeutic effect to be achieved, and (b) the limitationinherent in the art of compounding such active materials for thetreatment of disease in living subjects having a diseased condition inwhich bodily health is impaired as herein disclosed in detail.

The principal active ingredients are compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit parenteral dosage form can, for example, contain theprincipal active compound, i.e. an inhibitor, in amounts ranging fromabout 0.5 to about 100 mg, with from about 0.1 to 50 mg being preferred.The daily parenteral doses for mammalian subjects to be treated rangesfrom 0.01 mg/kg to 10 mg/kg of the inhibitor. The preferred daily dosagerange is 0.1 mg/kg to 1.0 mg/kg.

For oral dosages, the daily amount may range from 0.01 mg of activecompound/kg of mammalian subject to 100 mg/kg, preferably 0.1 to 10mg/kg of subject.

The inhibitor described above may form commonly known, pharmaceuticallyacceptable salts such as alkali metal and other common basic salts oracid addition salts, etc. References to the base substances aretherefore intended to include those common salts known to besubstantially equivalent to the parent compound and hydrates thereof.

The active compounds described herein are capable of further formingboth pharmaceutically acceptable acid addition and/or base salts. All ofthese forms are within the scope of the present invention.

Pharmaceutically acceptable acid addition salts of the active compoundsinclude salts derived from nontoxic inorganic acids such ashydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic,hydrofluoric, phosphorous, and the like, as well as the salts derivedfrom nontoxic organic acids, such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonicacids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate,sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate,oxalate, malonate succinate, suberate, sebacate, fumarate, maleate,mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate,lactate, maleate, tartrate, methanesulfonate, and the like. Alsocontemplated are salts of amino acids such as arginate and the like andgluconate, galacturonate (see, for example, Berge, S. M. et al,"Pharmaceutical Salts", JOURNAL OF PHARMACEUTICAL SCIENCE, 66, pp. 1-19(1977)).

The acid addition salts of said basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. Preferably, anactive compound can be converted to an acidic salt by treating with anaqueous solution of the desired acid, such that the resulting pH is lessthan 4. The solution can be passed through a C18 cartridge to absorb thecompound, washed with copious amounts of water, the compound eluted witha polar organic solvent such as, for example, methanol, acetonitrile,and the like, and isolated by concentrating under reduced pressurefollowed by lyophilization. The free base form may be regenerated bycontacting the salt form with a base and isolating the free base in theconventional manner. The free base forms differ from their respectivesalt forms somewhat in certain physical properties such as solubility inpolar solvents, but otherwise the salts are equivalent to theirrespective free base for purposes of the present invention.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge, S. M. et al, "Pharmaceutical Salts", JOURNALOF PHARMACEUTICAL SCIENCE, 66, pp. 1-19 (1977)).

The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. Preferably, anactive compound can be converted to a base salt by treating with anaqueous solution of the desired base, such that the resulting pH isgreater than 9. The solution can be passed through a C18 cartridge toabsorb the compound, washed with copious amounts of water, the compoundeluted with a polar organic solvent such as, for example, methanol,acetonitrile and the like, and isolated by concentrating under reducedpressure followed by lyophilization. The free acid form may beregenerated by contacting the salt form with an acid and isolating thefree acid in the conventional manner. The free acid forms differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents, but otherwise the salts are equivalentto their respective free acids for purposes of the present invention.

Certain of the compounds of the present invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral. the solvated forms, including hydrated forms are equivalent tounsolvated forms and are intended to be encompassed within the scope ofthe present invention.

Certain of the compounds of the present invention possess one or morechiral centers and such center may exist in the R(D) or S(L)configuration. The present invention includes all enantiomeric andepimeric forms as well as the appropriate mixtures thereof.

While the forms of the invention herein constitute presently preferredembodiments, many others are possible. It is not intended herein tomention all of the possible equivalent forms or ramifications of theinvention. It is understood that the terms used herein are merelydescriptive rather than limiting and that various changes may be madewithout departing from the spirit or scope of the invention. ##STR16##

What is claimed is:
 1. A compound of Formula Ia: wherein X is O, S, NH or NR⁷, wherein R⁷ is selected from the group consisting of lower alkyl (1-4 carbon atoms), OH, NH₂, lower alkoxy (1-4 carbon atoms) and lower monoalkylamino (1-4 carbon atoms);n is 0, 1 or 2; R¹ is H or lower alkyl (1-4 carbon atoms); if n is 2, R¹ is independently H or lower alkyl (1-4 carbon atoms) on either linking carbon atom; R² is selected from the group consisting of lower alkyl (1-4 carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms), nitro, halo (fluoro, chloro, bromo, iodo), lower perfluoroalkyl (1-4 carbon atoms), hydroxy, lower acyloxy (1-4 carbon atoms; --O--C(O)--R), amino, lower mono or dialkylamino (1-4 carbon atoms), lower mono or dicycloalkylamino (3-8 carbon atoms), hydroxymethyl, lower acyl (1-4 carbon atoms; --C(O)R), cyano, lower thioalkyl (1-4 carbon atoms), lower sulfinylalkyl (1-4 carbon atoms), lower sulfonylalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbon atoms), sulfinylcycloalkyl (3-8 carbon atoms), sulfonylcycloalkyl (3-8 carbon atoms), sulfonamido, lower mono or dialkylsulfonamido (1-4 carbon atoms), mono or dicycloalkylsulfonamido (3-8 carbon atoms), mercapto, carboxy, carboxamido (--C(O)--NH₂), lower mono or dialkylcarboxamido (1-4 carbon atoms), mono or dicycloalkoxycarboxamido (3-8 carbon atoms), lower alkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbon atoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbon atoms), lower alkynyl (2-4 carbon atoms), and two R² taken together on contiguous carbon atoms form a carbocyclic ring of 5-7 members or a monounsaturated ring selected from the group consisting of 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl, furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl, diazolanyl, piperazinyl, morpholino and thiomorpholino; m=0-3; Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl, pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl and quinazolinyl; R³, R⁴, and R⁶ are independently selected from the group consisting of H, lower alkyl (1-4 carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms), hydroxy, lower acyloxy (1-4 carbon atoms), amino, lower mono or dialkylamino (1-4 carbon atoms), lower mono or dicycloalkylamino (3-8 carbon atoms), carbonato (--OC(O)OR) where the R is lower alkyl of 1 to 4 carbon atoms or cycloalkyl of 3-8 carbon atoms; ureido or thioureido or N- or O- linked urethane any one of which is optionally substituted by mono or di-lower alkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms); lower thioalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbon atoms), mercapto, lower alkenyl (2-4 carbon atoms), hydrazino, N'- lower alkylhydrazino (1-4 carbon atoms), lower acylamino (1-4 carbon atoms), hydroxylamino, and lower O- alkylhydroxylamino (1-4 carbon atoms); or optionally R³ and R⁴ or R⁴ and R⁶ taken together on contiguous carbon atoms form a carbocyclic ring of 5-7 members or a monounsaturated ring selected from the group consisting of 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl, furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl, diazolanyl, piperazinyl, morpholino and thiomorpholino; any lower alkyl group substituent on any of the substituents in R³, R⁴ and R⁶ which moiety is optionally substituted with one or more groups selected from the group consisting of hydroxy, amino, lower monoalkylamino, lower dialkylamino, N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino, N-thiomorpholino and N-piperazino; with the proviso that R³ cannot be either OH or SH; with the further proviso that at least one of the R³ and R⁴ or R⁴ and R⁶ substituents must be other than hydrogen, halo, lower alkyl (1-4 carbon atoms), or lower alkoxy (1-4 carbon atoms); optionally if any of the substituents R¹, R², R³, R⁴, or R⁶ have chiral centers, then all stereoisomers thereof both separately and as racemic and/or diastereoisomeric mixtures are included; or a pharmaceutical salt or hydrate thereof.
 2. The compound of claim 1 wherein the compound is selected from the group consisting of 6-Amino-4-(3-bromoanilino) pyrido[3,2-d]pyrimidine; 4-(3-Bromoanilino)-6-methylaminopyrido [3,2-d]pyrimidine; and 4-(3-Bromoanilino)-6-methylaminopyrido[3,2-d]pyrimidine. 